Science.gov

Sample records for expressions optical microscopy

  1. Pure optical photoacoustic microscopy

    PubMed Central

    Xie, Zhixing; Chen, Sung-Liang; Ling, Tao; Guo, L. Jay; Carson, Paul L.; Wang, Xueding

    2011-01-01

    The concept of pure optical photoacoustic microscopy(POPAM) was proposed based on optical rastering of a focused excitation beam and optically sensing the photoacoustic signal using a microring resonator fabricated by a nanoimprinting technique. After the refinements of the microring’s working wavelength and in the resonator structure and mold fabrication, an ultrahigh Q factor of 3.0×105 was achieved which provided high sensitivity with a noise equivalent detectable pressure(NEDP) value of 29Pa. This NEDP is much lower than the hundreds of Pascals achieved with existing optical resonant structures such as etalons, fiber gratings and dielectric multilayer interference filters available for acoustic measurement. The featured high sensitivity allowed the microring resonator to detect the weak photoacoustic signals from micro- or submicroscale objects. The inherent superbroad bandwidth of the optical microring resonator combined with an optically focused scanning beam provided POPAM with high resolution in the axial as well as both lateral directions while the axial resolution of conventional photoacoustic microscopy (PAM) suffers from the limited bandwidth of PZT detectors. Furthermore, the broadband microring resonator showed similar sensitivity to that of our most sensitive PZT detector. The current POPAM system provides a lateral resolution of 5 μm and an axial resolution of 8 μm, comparable to that achieved by optical microscopy while presenting the unique contrast of optical absorption and functional information complementing other optical modalities. The 3D structure of microvasculature, including capillary networks, and even individual red blood cells have been discerned successfully in the proof-of-concept experiments on mouse bladders ex vivo and mouse ears in vivo. The potential of approximately GHz bandwidth of the microring resonator also might allow much higher resolution than shown here in microscopy of optical absorption and acoustic propagation

  2. Multimodal Nonlinear Optical Microscopy

    PubMed Central

    Yue, Shuhua; Slipchenko, Mikhail N.; Cheng, Ji-Xin

    2013-01-01

    Because each nonlinear optical (NLO) imaging modality is sensitive to specific molecules or structures, multimodal NLO imaging capitalizes the potential of NLO microscopy for studies of complex biological tissues. The coupling of multiphoton fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering (CARS) has allowed investigation of a broad range of biological questions concerning lipid metabolism, cancer development, cardiovascular disease, and skin biology. Moreover, recent research shows the great potential of using CARS microscope as a platform to develop more advanced NLO modalities such as electronic-resonance-enhanced four-wave mixing, stimulated Raman scattering, and pump-probe microscopy. This article reviews the various approaches developed for realization of multimodal NLO imaging as well as developments of new NLO modalities on a CARS microscope. Applications to various aspects of biological and biomedical research are discussed. PMID:24353747

  3. Visual-servoing optical microscopy

    DOEpatents

    Callahan, Daniel E.; Parvin, Bahram

    2011-05-24

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

  4. Visual-servoing optical microscopy

    DOEpatents

    Callahan, Daniel E.; Parvin, Bahram

    2009-06-09

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

  5. Visual-servoing optical microscopy

    DOEpatents

    Callahan, Daniel E; Parvin, Bahram

    2013-10-01

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

  6. Developments in optical coherence microscopy

    NASA Astrophysics Data System (ADS)

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

    2010-11-01

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

  7. Gabor domain optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Murali, Supraja

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

  8. Scanning Tunneling Optical Resonance Microscopy

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  9. Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

    PubMed Central

    Kim, Doory; Deerinck, Thomas J.; Sigal, Yaron M.; Babcock, Hazen P.; Ellisman, Mark H.; Zhuang, Xiaowei

    2015-01-01

    Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets. PMID:25874453

  10. Disposable optics for microscopy diagnostics.

    PubMed

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

    2015-01-01

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

  11. Disposable optics for microscopy diagnostics

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  12. Disposable optics for microscopy diagnostics

    PubMed Central

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

    2015-01-01

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

  13. Optical microscopy versus scanning electron microscopy in urolithiasis.

    PubMed

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

    2009-10-01

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

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

    SciTech Connect

    Ramanathan, Nathan Muruganathan; Darling, Seth B.

    2015-01-01

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

  15. Tomographic phase microscopy using optical tweezers

    NASA Astrophysics Data System (ADS)

    Habaza, Mor; Gilboa, Barak; Roichman, Yael; Shaked, Natan T.

    2015-07-01

    We review our technique for tomographic phase microscopy with optical tweezers [1]. This tomographic phase microscopy approach enables full 3-D refractive-index reconstruction. Tomographic phase microscopy measures quantitatively the 3- D distribution of refractive-index in biological cells. We integrated our external interferometric module with holographic optical tweezers for obtaining quantitative phase maps of biological samples from a wide range of angles. The close-tocommon- path, off-axis interferometric system enables a full-rotation tomographic acquisition of a single cell using holographic optical tweezers for trapping and manipulating with a desired array of traps, while acquiring phase information of a single cell from all different angles and maintaining the native surrounding medium. We experimentally demonstrated two reconstruction algorithms: the filtered back-projection method and the Fourier diffraction method for 3-D refractive index imaging of yeast cells.

  16. Subwavelength optical microscopy in the far field

    SciTech Connect

    Sun Qingqing; Zubairy, M. Suhail; Al-Amri, M.; Scully, Marlan O.

    2011-06-15

    We present a procedure for subwavelength optical microscopy. The identical atoms are distributed on a plane and shined with a standing wave. We rotate the plane to different angles and record the resonant fluorescence spectra in the far field, from which we can obtain their distance and location information. This procedure also works for atomic separation above one wavelength and therefore provides a seamless microscopy.

  17. Optical Property Analyses of Plant Cells for Adaptive Optics Microscopy

    NASA Astrophysics Data System (ADS)

    Tamada, Yosuke; Murata, Takashi; Hattori, Masayuki; Oya, Shin; Hayano, Yutaka; Kamei, Yasuhiro; Hasebe, Mitsuyasu

    2014-04-01

    In astronomy, adaptive optics (AO) can be used to cancel aberrations caused by atmospheric turbulence and to perform diffraction-limited observation of astronomical objects from the ground. AO can also be applied to microscopy, to cancel aberrations caused by cellular structures and to perform high-resolution live imaging. As a step toward the application of AO to microscopy, here we analyzed the optical properties of plant cells. We used leaves of the moss Physcomitrella patens, which have a single layer of cells and are thus suitable for optical analysis. Observation of the cells with bright field and phase contrast microscopy, and image degradation analysis using fluorescent beads demonstrated that chloroplasts provide the main source of optical degradations. Unexpectedly, the cell wall, which was thought to be a major obstacle, has only a minor effect. Such information provides the basis for the application of AO to microscopy for the observation of plant cells.

  18. Optical microscopy beyond the diffraction limit

    PubMed Central

    Smolyaninov, Igor I.

    2008-01-01

    Over the past century the resolution of far-field optical microscopes, which rely on propagating optical modes, was widely believed to be limited because of diffraction to a value on the order of a half-wavelength λ∕2 of the light used. Although immersion microscopes had slightly improved resolution on the order of λ∕2n, the increased resolution was limited by the small range of refractive indices, n, of available transparent materials. We are experiencing quick demolition of the diffraction limit in optical microscopy. Over the past few years numerous nonlinear optical microscopy techniques based on photoswitching and saturation of fluorescence demonstrated far-field resolution of 20 to 30 nm. The latest exciting example of these techniques has been demonstrated by Huang et al. [Science 319, 810–813 (2008)]. Moreover, recent progress in metamaterials indicates that artificial optical media can be created, which do not exhibit the diffraction limit. Resolution of linear “immersion” microscopes based on such metamaterials appears limited only by losses, which can be compensated by gain media. Thus, optical microscopy is quickly moving towards the 10 nm resolution scale, which should bring about numerous revolutionary advances in biomedical imaging. PMID:19404465

  19. Coherent nonlinear optical imaging: beyond fluorescence microscopy.

    PubMed

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

    2011-01-01

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

  20. Coherent Nonlinear Optical Imaging: Beyond Fluorescence Microscopy

    PubMed Central

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

    2012-01-01

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

  1. Near-Field Scanning Optical Microscopy and Raman Microscopy.

    NASA Astrophysics Data System (ADS)

    Harootunian, Alec Tate

    1987-09-01

    Both a one dimensional near-field scanning optical microscope and Raman microprobe were constructed. In near -field scanning optical microscopy (NSOM) a subwavelength aperture is scanned in the near-field of the object. Radiation transmitted through the aperture is collected to form an image as the aperture scans over the object. The resolution of an NSOM system is essentially wavelength independent and is limited by the diameter of the aperture used to scan the object. NSOM was developed in an effort to provide a nondestructive in situ high spatial resolution probe while still utilizing photons at optical wavelengths. The Raman microprobe constructed provided vibrational Raman information with spatial resolution equivalent that of a conventional diffraction limited microscope. Both transmission studies and near-field diffration studies of subwavelength apertures were performed. Diffraction theories for a small aperture in an infinitely thin conducting screen, a slit in a thick conducting screen, and an aperture in a black screen were examined. All three theories indicate collimation of radiation to the size to the size of the subwavelength aperture or slit in the near-field. Theoretical calculations and experimental results indicate that light transmitted through subwavelength apertures is readily detectable. Light of wavelength 4579 (ANGSTROM) was transmitted through apertures with diameters as small as 300 (ANGSTROM). These studies indicate the feasibility of constructing an NSOM system. One dimensional transmission and fluorescence NSOM systems were constructed. Apertures in the tips of metallized glass pipettes width inner diameters of less than 1000 (ANGSTROM) were used as a light source in the NSOM system. A tunneling current was used to maintain the aperture position in the near-field. Fluorescence NSOM was demonstrated for the first time. Microspectroscopic and Raman microscopic studies of turtle cone oil droplets were performed. Both the Raman vibrational

  2. Hyperlens-array-implemented optical microscopy

    NASA Astrophysics Data System (ADS)

    Iwanaga, Masanobu

    2014-08-01

    Limit of resolution of conventional optical microscopes has never reached below 100 nm under visible light illumination. We show that numerically designed high-transmittance hyperlens array (HLA) is implemented in an optical microscope and works in practice for achieving one-shot-recording optical images of in-situ placed objects with sub 50 nm resolution in lateral direction. Direct resolution test employing well-defined nanopatterns proves that the HLA-implemented imaging is super-resolution optical microscopy, which works even under nW/mm2 visible illumination for objects. The HLA implementation makes the resolution of conventional microscopes one-scale higher, leading to the 1/10 illumination wavelength range, that is, mesoscopic range.

  3. Virtual k -Space Modulation Optical Microscopy

    NASA Astrophysics Data System (ADS)

    Kuang, Cuifang; Ma, Ye; Zhou, Renjie; Zheng, Guoan; Fang, Yue; Xu, Yingke; Liu, Xu; So, Peter T. C.

    2016-07-01

    We report a novel superresolution microscopy approach for imaging fluorescence samples. The reported approach, termed virtual k -space modulation optical microscopy (VIKMOM), is able to improve the lateral resolution by a factor of 2, reduce the background level, improve the optical sectioning effect and correct for unknown optical aberrations. In the acquisition process of VIKMOM, we used a scanning confocal microscope setup with a 2D detector array to capture sample information at each scanned x -y position. In the recovery process of VIKMOM, we first modulated the captured data by virtual k -space coding and then employed a ptychography-inspired procedure to recover the sample information and correct for unknown optical aberrations. We demonstrated the performance of the reported approach by imaging fluorescent beads, fixed bovine pulmonary artery endothelial (BPAE) cells, and living human astrocytes (HA). As the VIKMOM approach is fully compatible with conventional confocal microscope setups, it may provide a turn-key solution for imaging biological samples with ˜100 nm lateral resolution, in two or three dimensions, with improved optical sectioning capabilities and aberration correcting.

  4. Virtual k-Space Modulation Optical Microscopy.

    PubMed

    Kuang, Cuifang; Ma, Ye; Zhou, Renjie; Zheng, Guoan; Fang, Yue; Xu, Yingke; Liu, Xu; So, Peter T C

    2016-07-01

    We report a novel superresolution microscopy approach for imaging fluorescence samples. The reported approach, termed virtual k-space modulation optical microscopy (VIKMOM), is able to improve the lateral resolution by a factor of 2, reduce the background level, improve the optical sectioning effect and correct for unknown optical aberrations. In the acquisition process of VIKMOM, we used a scanning confocal microscope setup with a 2D detector array to capture sample information at each scanned x-y position. In the recovery process of VIKMOM, we first modulated the captured data by virtual k-space coding and then employed a ptychography-inspired procedure to recover the sample information and correct for unknown optical aberrations. We demonstrated the performance of the reported approach by imaging fluorescent beads, fixed bovine pulmonary artery endothelial (BPAE) cells, and living human astrocytes (HA). As the VIKMOM approach is fully compatible with conventional confocal microscope setups, it may provide a turn-key solution for imaging biological samples with ∼100  nm lateral resolution, in two or three dimensions, with improved optical sectioning capabilities and aberration correcting. PMID:27447529

  5. Pulse front adaptive optics in multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Sun, B.; Salter, P. S.; Booth, M. J.

    2016-03-01

    The accurate focusing of ultrashort laser pulses is extremely important in multiphoton microscopy. Using adaptive optics to manipulate the incident ultrafast beam in either the spectral or spatial domain can introduce significant benefits when imaging. Here we introduce pulse front adaptive optics: manipulating an ultrashort pulse in both the spatial and temporal domains. A deformable mirror and a spatial light modulator are operated in concert to modify contours of constant intensity in space and time within an ultrashort pulse. Through adaptive control of the pulse front, we demonstrate an enhancement in the measured fluorescence from a two photon microscope.

  6. Multiple scattering in optical coherence microscopy.

    PubMed

    Yadlowsky, M J; Schmitt, J M; Bonner, R F

    1995-09-01

    We show that the multiple-scatter rejection provided by optical coherence microscopy (low-coherence interferometry) can be incomplete in optically turbid media and that multiple scattering manifests itself in two distinct ways. Multiple small-angle scattering results in an effective probe field that is stronger than expected from a first-order beam extinction model, but that contains a distorted wave front that enhances the apparent reflectance of small structures relative to those that are larger than the unscattered incident beam. Multiple wide-angle scattering produces a broad diffuse haze that reduces the contrast of subsequent features. PMID:21060400

  7. Near-field optical microscopy nanoarray

    NASA Astrophysics Data System (ADS)

    Semin, David J.; Ambrose, W. Patrick; Goodwin, Peter M.; Wendt, Joel R.; Keller, Richard A.

    1997-04-01

    Multiplexing near-field scanning optical microscopy (NSOM) by the use of a nanoarray with parallel imaging is studied. The fabrication, characterization, and utilization of nanoarrays with approximately 100 nm diameter apertures spaced 500 nm center-to-center is presented. Extremely uniform nanoarrays with approximately 108 apertures were fabricated by electron beam lithography and reactive ion etching. The nanoarrays were characterized by atomic force microscopy and scanning electron microscopy. In this paper we utilize these nanoarrays in a laser-illuminated microscope with parallel detection on a charge-coupled device. Detection of B-phycoerythrin molecules using near- field illumination is presented. In principle, our system can be used to obtain high lateral resolution NSOM images over a wide-field of view (e.g. 50 - 100 micrometers ) within seconds.

  8. A near-field optical microscopy nanoarray

    SciTech Connect

    Semin, D.J.; Ambrose, W.P.; Goodwin, P.M.; Kwller, A.; Wendt, J.R.

    1996-12-31

    Multiplexing near-field scanning optical microscopy (NSOM) by the use of a nanoarray with parallel imaging is studied. The fabrication, characterization, and utilization of nanoarrays with {approximately} 100 nm diameter apertures spaced 500 nm center-to- center is presented. Extremely uniform nanoarrays with {approximately} 10{sup 8} apertures were fabricated by electron beam lithography and reactive ion etching. The nanoarrays were characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). In this paper we utilize these nanoarrays in a laser-illuminated microscope with parallel detection on a charge- coupled device (CCD). Detection of B-phycoerythrin (B-PE) molecules using near-field illumination is presented. In principle, our system can be used to obtain high lateral resolution NSOM images over a wide-field of view (e.g. 50-100 {mu}m) within seconds.

  9. Dark-field optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Pache, C.; Villiger, M. L.; Lasser, T.

    2010-02-01

    Many solutions have been proposed to produce phase quantitative images of biological cell samples. Among these, Spectral Domain Phase Microscopy combines the fast imaging speed and high sensitivity of Optical Coherence Microscopy (OCM) in the Fourier domain with the high phase stability of common-path interferometry. We report on a new illumination scheme for OCM that enhances the sensitivity for backscattered light and detects the weak sample signal, otherwise buried by the signal from specular reflection. With the use of a Bessel-like beam, a dark-field configuration was realized. Sensitivity measurements for three different illumination configurations were performed to compare our method to standard OCM and extended focus OCM. Using a well-defined scattering and reflecting object, we demonstrated an attenuation of -40 dB of the DC-component and a relative gain of 30 dB for scattered light, compared to standard OCM. In a second step, we applied this technique, referred to as dark-field Optical Coherence Microscopy (dfOCM), to living cells. Chinese hamster ovarian cells were applied in a drop of medium on a coverslide. The cells of ~15 μm in diameter and even internal cell structures were visualized in the acquired tomograms.

  10. Single-spin stochastic optical reconstruction microscopy

    PubMed Central

    Pfender, Matthias; Aslam, Nabeel; Waldherr, Gerald; Neumann, Philipp; Wrachtrup, Jörg

    2014-01-01

    We experimentally demonstrate precision addressing of single-quantum emitters by combined optical microscopy and spin resonance techniques. To this end, we use nitrogen vacancy (NV) color centers in diamond confined within a few ten nanometers as individually resolvable quantum systems. By developing a stochastic optical reconstruction microscopy (STORM) technique for NV centers, we are able to simultaneously perform sub–diffraction-limit imaging and optically detected spin resonance (ODMR) measurements on NV spins. This allows the assignment of spin resonance spectra to individual NV center locations with nanometer-scale resolution and thus further improves spatial discrimination. For example, we resolved formerly indistinguishable emitters by their spectra. Furthermore, ODMR spectra contain metrology information allowing for sub–diffraction-limit sensing of, for instance, magnetic or electric fields with inherently parallel data acquisition. As an example, we have detected nuclear spins with nanometer-scale precision. Finally, we give prospects of how this technique can evolve into a fully parallel quantum sensor for nanometer resolution imaging of delocalized quantum correlations. PMID:25267655

  11. Multiparallel Three-Dimensional Optical Microscopy

    NASA Technical Reports Server (NTRS)

    Nguyen, Lam K.; Price, Jeffrey H.; Kellner, Albert L.; Bravo-Zanoquera, Miguel

    2010-01-01

    Multiparallel three-dimensional optical microscopy is a method of forming an approximate three-dimensional image of a microscope sample as a collection of images from different depths through the sample. The imaging apparatus includes a single microscope plus an assembly of beam splitters and mirrors that divide the output of the microscope into multiple channels. An imaging array of photodetectors in each channel is located at a different distance along the optical path from the microscope, corresponding to a focal plane at a different depth within the sample. The optical path leading to each photodetector array also includes lenses to compensate for the variation of magnification with distance so that the images ultimately formed on all the photodetector arrays are of the same magnification. The use of optical components common to multiple channels in a simple geometry makes it possible to obtain high light-transmission efficiency with an optically and mechanically simple assembly. In addition, because images can be read out simultaneously from all the photodetector arrays, the apparatus can support three-dimensional imaging at a high scanning rate.

  12. Digital holographic microscopy combined with optical tweezers

    NASA Astrophysics Data System (ADS)

    Cardenas, Nelson; Yu, Lingfeng; Mohanty, Samarendra K.

    2011-02-01

    While optical tweezers have been widely used for the manipulation and organization of microscopic objects in three dimensions, observing the manipulated objects along axial direction has been quite challenging. In order to visualize organization and orientation of objects along axial direction, we report development of a Digital holographic microscopy combined with optical tweezers. Digital holography is achieved by use of a modified Mach-Zehnder interferometer with digital recording of interference pattern of the reference and sample laser beams by use of a single CCD camera. In this method, quantitative phase information is retrieved dynamically with high temporal resolution, only limited by frame rate of the CCD. Digital focusing, phase-unwrapping as well as online analysis and display of the quantitative phase images was performed on a software developed on LabView platform. Since phase changes observed in DHOT is very sensitive to optical thickness of trapped volume, estimation of number of particles trapped in the axial direction as well as orientation of non-spherical objects could be achieved with high precision. Since in diseases such as malaria and diabetics, change in refractive index of red blood cells occurs, this system can be employed to map such disease-specific changes in biological samples upon immobilization with optical tweezers.

  13. Scanning Tunneling Optical Resonance Microscopy Developed

    NASA Technical Reports Server (NTRS)

    Bailey, Sheila G.; Raffaelle, Ryne P.; Lau, Janis E.; Jenkins, Phillip P.; Castro, Stephanie L.; Tin, Padetha; Wilt, David M.; Pal, Anna Maria; Fahey, Stephen D.

    2004-01-01

    The ability to determine the in situ optoelectronic properties of semiconductor materials has become especially important as the size of device architectures has decreased and the development of complex microsystems has increased. Scanning Tunneling Optical Resonance Microscopy, or STORM, can interrogate the optical bandgap as a function of its position within a semiconductor micro-structure. This technique uses a tunable solidstate titanium-sapphire laser whose output is "chopped" using a spatial light modulator and is coupled by a fiber-optic connector to a scanning tunneling microscope in order to illuminate the tip-sample junction. The photoenhanced portion of the tunneling current is spectroscopically measured using a lock-in technique. The capabilities of this technique were verified using semiconductor microstructure calibration standards that were grown by organometallic vapor-phase epitaxy. Bandgaps characterized by STORM measurements were found to be in good agreement with the bulk values determined by transmission spectroscopy and photoluminescence and with the theoretical values that were based on x-ray diffraction results.

  14. Multifocal multiphoton microscopy with adaptive optical correction

    NASA Astrophysics Data System (ADS)

    Coelho, Simao; Poland, Simon; Krstajic, Nikola; Li, David; Monypenny, James; Walker, Richard; Tyndall, David; Ng, Tony; Henderson, Robert; Ameer-Beg, Simon

    2013-02-01

    Fluorescence lifetime imaging microscopy (FLIM) is a well established approach for measuring dynamic signalling events inside living cells, including detection of protein-protein interactions. The improvement in optical penetration of infrared light compared with linear excitation due to Rayleigh scattering and low absorption have provided imaging depths of up to 1mm in brain tissue but significant image degradation occurs as samples distort (aberrate) the infrared excitation beam. Multiphoton time-correlated single photon counting (TCSPC) FLIM is a method for obtaining functional, high resolution images of biological structures. In order to achieve good statistical accuracy TCSPC typically requires long acquisition times. We report the development of a multifocal multiphoton microscope (MMM), titled MegaFLI. Beam parallelization performed via a 3D Gerchberg-Saxton (GS) algorithm using a Spatial Light Modulator (SLM), increases TCSPC count rate proportional to the number of beamlets produced. A weighted 3D GS algorithm is employed to improve homogeneity. An added benefit is the implementation of flexible and adaptive optical correction. Adaptive optics performed by means of Zernike polynomials are used to correct for system induced aberrations. Here we present results with significant improvement in throughput obtained using a novel complementary metal-oxide-semiconductor (CMOS) 1024 pixel single-photon avalanche diode (SPAD) array, opening the way to truly high-throughput FLIM.

  15. Spectral fusing Gabor domain optical coherence microscopy.

    PubMed

    Meemon, Panomsak; Widjaja, Joewono; Rolland, Jannick P

    2016-02-01

    Gabor domain optical coherence microscopy (GD-OCM) is one of many variations of optical coherence tomography (OCT) techniques that aims for invariant high resolution across a 3D field of view by utilizing the ability to dynamically refocus the imaging optics in the sample arm. GD-OCM acquires multiple cross-sectional images at different focus positions of the objective lens, and then fuses them to obtain an invariant high-resolution 3D image of the sample, which comes with the intrinsic drawback of a longer processing time as compared to conventional Fourier domain OCT. Here, we report on an alternative Gabor fusing algorithm, the spectral-fusion technique, which directly processes each acquired spectrum and combines them prior to the Fourier transformation to obtain a depth profile. The implementation of the spectral-fusion algorithm is presented and its performance is compared to that of the prior GD-OCM spatial-fusion approach. The spectral-fusion approach shows twice the speed of the spatial-fusion approach for a spectrum size of less than 2000 point sampling, which is a commonly used spectrum size in OCT imaging, including GD-OCM. PMID:26907410

  16. Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM.

    PubMed

    Hirano, Kazumi; Kinoshita, Takaaki; Uemura, Takeshi; Motohashi, Hozumi; Watanabe, Yohei; Ebihara, Tatsuhiko; Nishiyama, Hidetoshi; Sato, Mari; Suga, Mitsuo; Maruyama, Yuusuke; Tsuji, Noriko M; Yamamoto, Masayuki; Nishihara, Shoko; Sato, Chikara

    2014-08-01

    Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates high-throughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment. Neuron differentiation, neural networking, proplatelet-formation and phagocytosis were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. Fas expression on the cell surface was visualized, correlated to the spatial distribution of F-actin. Axonal partitioning was studied using primary-culture neurons, and presynaptic induction by GluRδ2-N-terminus-linked fluorescent magnetic beads was correlated to the presynaptic-marker Bassoon. Further, megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by dendritic cells was also imaged. Based on these studies, ASEM correlative microscopy promises to allow the study of various mesoscopic-scale dynamics in the near future. PMID:24216127

  17. Improved optical fiber probes for scanning near field optical microscopy

    NASA Astrophysics Data System (ADS)

    Wheaton, Bryan R.

    2004-12-01

    The motivation behind this work stems from a combination of my interest in atomic force microscopy (AFM) and the need to apply AFM to several areas of glass research. AFM was used as the main characterization tool in the study of near-field scanning optical microscopy (NSOM) tip formation, evaluation of phase separation in glasses and copper oxide semiconductor film formation. The use of atomic force microscopy (AFM) to evaluate the evolving tip structure of an optical fiber probe for NSOM was studied. This study demonstrates the feasibility of predicting the final tip cone angle, without taking the etching process to completion. Cone angles reported in this study ranged from 58 to 152 degrees, depending on the fiber type and etch conditions. The ability to vary the probe cone angle, and utilize AFM to evaluate the cone angle that results from a set of etch conditions, are valuable additions to the development of NSOM fiber tips. The chemical and spatial variation of phase separated morphologies in glasses can range from a few angstroms to microns, often requiring very high magnification for detection. Historically phase separated glasses have been characterized by transmission electron microscopy (TEM), a time consuming and costly technique. Atomic force microscopy (AFM) provides an inexpensive alternative to TEM and has proven to be a powerful tool in the evaluation of type, degree and scale of phase separation in glasses down to the nanometer level. AFM was used to show that the thickness and uniformity of the CuO films grown in-situ on the surface of copper containing alkali borosilicate glasses increased with time and temperature, however an upper time limit was reached in which no further thickness increases were realized. Tenorite, cuprite and copper metal films were produced depending on the heat treatment environment. XPS was utilized to confirm that copper oxide film formation during heat treatments of glasses near Tg results from the oxidation of copper

  18. Brillouin Optical Microscopy for Corneal Biomechanics

    PubMed Central

    Scarcelli, Giuliano; Pineda, Roberto

    2012-01-01

    Purpose. The mechanical properties of corneal tissue are linked to prevalent ocular diseases and therapeutic procedures. Brillouin microscopy is a novel optical technology that enables three-dimensional mechanical imaging. In this study, the feasibility of this noncontact technique was tested for in situ quantitative assessment of the biomechanical properties of the cornea. Methods. Brillouin light-scattering involves a spectral shift proportional to the longitudinal modulus of elasticity of the tissue. A 532-nm single-frequency laser and a custom-developed ultrahigh-resolution spectrometer were used to measure the Brillouin frequency. Confocal scanning was used to perform Brillouin elasticity imaging of the corneas of whole bovine eyes. The longitudinal modulus of the bovine corneas was compared before and after riboflavin corneal collagen photo-cross-linking. The Brillouin measurements were then compared with conventional stress–strain mechanical test results. Results. High-resolution Brillouin images of the cornea were obtained, revealing a striking depth-dependent variation of the elastic modulus across the cornea. Along the central axis, the Brillouin frequency shift varied gradually from 8.2 GHz in the epithelium to 7.5 GHz near the endothelium. The coefficients of the down slope were measured to be approximately 1.09, 0.32, and 2.94 GHz/mm in the anterior, posterior, and innermost stroma, respectively. On riboflavin collagen cross-linking, marked changes in the axial Brillouin profiles (P < 0.001) were noted before and after cross-linking. Conclusions. Brillouin imaging can assess the biomechanical properties of cornea in situ with high spatial resolution. This novel technique has the potential for use in clinical diagnostics and treatment monitoring. PMID:22159012

  19. Tunable thin-film optical filters for hyperspectral microscopy

    NASA Astrophysics Data System (ADS)

    Favreau, Peter F.; Rich, Thomas C.; Prabhat, Prashant; Leavesley, Silas J.

    2013-02-01

    Hyperspectral imaging was originally developed for use in remote sensing applications. More recently, it has been applied to biological imaging systems, such as fluorescence microscopes. The ability to distinguish molecules based on spectral differences has been especially advantageous for identifying fluorophores in highly autofluorescent tissues. A key component of hyperspectral imaging systems is wavelength filtering. Each filtering technology used for hyperspectral imaging has corresponding advantages and disadvantages. Recently, a new optical filtering technology has been developed that uses multi-layered thin-film optical filters that can be rotated, with respect to incident light, to control the center wavelength of the pass-band. Compared to the majority of tunable filter technologies, these filters have superior optical performance including greater than 90% transmission, steep spectral edges and high out-of-band blocking. Hence, tunable thin-film optical filters present optical characteristics that may make them well-suited for many biological spectral imaging applications. An array of tunable thin-film filters was implemented on an inverted fluorescence microscope (TE 2000, Nikon Instruments) to cover the full visible wavelength range. Images of a previously published model, GFP-expressing endothelial cells in the lung, were acquired using a charge-coupled device camera (Rolera EM-C2, Q-Imaging). This model sample presents fluorescently-labeled cells in a highly autofluorescent environment. Linear unmixing of hyperspectral images indicates that thin-film tunable filters provide equivalent spectral discrimination to our previous acousto-optic tunable filter-based approach, with increased signal-to-noise characteristics. Hence, tunable multi-layered thin film optical filters may provide greatly improved spectral filtering characteristics and therefore enable wider acceptance of hyperspectral widefield microscopy.

  20. Brain plasticity and functionality explored by nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Sacconi, L.; Allegra, L.; Buffelli, M.; Cesare, P.; D'Angelo, E.; Gandolfi, D.; Grasselli, G.; Lotti, J.; Mapelli, J.; Strata, P.; Pavone, F. S.

    2010-02-01

    In combination with fluorescent protein (XFP) expression techniques, two-photon microscopy has become an indispensable tool to image cortical plasticity in living mice. In parallel to its application in imaging, multi-photon absorption has also been used as a tool for the dissection of single neurites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. In this work, multi-photon nanosurgery is applied to dissect single climbing fibers expressing GFP in the cerebellar cortex. The morphological consequences are then characterized with time lapse 3-dimensional two-photon imaging over a period of minutes to days after the procedure. Preliminary investigations show that the laser induced fiber dissection recalls a regenerative process in the fiber itself over a period of days. These results show the possibility of this innovative technique to investigate regenerative processes in adult brain. In parallel with imaging and manipulation technique, non-linear microscopy offers the opportunity to optically record electrical activity in intact neuronal networks. In this work, we combined the advantages of second-harmonic generation (SHG) with a random access (RA) excitation scheme to realize a new microscope (RASH) capable of optically recording fast membrane potential events occurring in a wide-field of view. The RASH microscope, in combination with bulk loading of tissue with FM4-64 dye, was used to simultaneously record electrical activity from clusters of Purkinje cells in acute cerebellar slices. Complex spikes, both synchronous and asynchronous, were optically recorded simultaneously across a given population of neurons. Spontaneous electrical activity was also monitored simultaneously in pairs of neurons, where action potentials were recorded without averaging across trials. These results show the strength of this technique in describing the temporal dynamics of neuronal assemblies, opening promising

  1. Ultrafast optical pulse delivery with fibers for nonlinear microscopy

    PubMed Central

    Kim, Daekeun; Choi, Heejin; Yazdanfar, Siavash; So, Peter T. C.

    2008-01-01

    Nonlinear microscopies including multiphoton excitation fluorescence microscopy and multiple-harmonic generation microscopy have recently gained popularity for cellular and tissue imaging. The optimization of these imaging methods for minimally invasive use will require optical fibers to conduct light into tight space where free space delivery is difficult. The delivery of high peak power laser pulses with optical fibers is limited by dispersion resulting from nonlinear refractive index responses. In this paper, we characterize a variety of commonly used optical fibers in terms of how they affect pulse profile and imaging performance of nonlinear microscopy; the following parameters are quantified: spectral bandwidth and temporal pulse width, two-photon excitation efficiency, and optical resolution. A theoretical explanation for the measured performance of these is also provided. PMID:18816597

  2. Nonlinear optical microscopy for imaging thin films and surfaces

    SciTech Connect

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

    1995-03-01

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

  3. Micromachined photoplastic probe for scanning near-field optical microscopy

    NASA Astrophysics Data System (ADS)

    Genolet, G.; Despont, M.; Vettiger, P.; Staufer, U.; Noell, W.; de Rooij, N. F.; Cueni, T.; Bernal, M.-P.; Marquis-Weible, F.

    2001-10-01

    We present a hybrid probe for scanning near-field optical microscopy (SNOM), which consists of a micromachined photoplastic tip with a metallic aperture at the apex that is attached to an optical fiber, thus combining the advantages of optical fiber probes and micromachined tips. The tip and aperture are batch fabricated and assembled to a preetched optical fiber with micrometer centering precision. Rectangular apertures of 50 nm×130 nm have been produced without the need of any postprocessing. Topographical and optical imaging with a probe having an aperture of 300 nm demonstrate the great potential of the photoplastic probe for SNOM applications.

  4. Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy

    SciTech Connect

    Miranda, Adelaide; De Beule, Pieter A. A.

    2015-09-15

    Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discuss sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate.

  5. Adaptive optics in digital micromirror based confocal microscopy

    NASA Astrophysics Data System (ADS)

    Pozzi, P.; Wilding, D.; Soloviev, O.; Vdovin, G.; Verhaegen, M.

    2016-03-01

    This proceeding reports early results in the development of a new technique for adaptive optics in confocal microscopy. The term adaptive optics refers to the branch of optics in which an active element in the optical system is used to correct inhomogeneities in the media through which light propagates. In its most classical form, mostly used in astronomical imaging, adaptive optics is achieved through a closed loop in which the actuators of a deformable mirror are driven by a wavefront sensor. This approach is severely limited in fluorescence microscopy, as the use of a wavefront sensor requires the presence of a bright, point like source in the field of view, a condition rarely satisfied in microscopy samples. Previously reported approaches to adaptive optics in fluorescence microscopy are therefore limited to the inclusion of fluorescent microspheres in the sample, to use as bright stars for wavefront sensors, or time consuming sensorless optimization procedures, requiring several seconds of optimization before the acquisition of a single image. We propose an alternative approach to the problem, implementing sensorless adaptive optics in a Programmable array microscope. A programmable array microscope is a microscope based on a digital micromirror device, in which the single elements of the micromirror act both as point sources and pinholes.

  6. Confocal device and application strategies for endoluminal optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    George, Markus; Schnieder, Ludger; Buess, Gerhard F.

    2003-10-01

    While endoscopic optical coherence tomography has been established successfully in vivo ,implementation of endoluminal optical coherence microscopy remains demanding,s suitable confocal probe is lacking. A miniaturized confocal laser scanning microscope is presented,which fulfills the requirements for endoluminal optical coherence microscopy. First,imaging experience gained for optical coherence microscopy of nimal gastrointestinal tissue samples is described. For this purpose,laboratory scale optical coherence microscope with an image acquisition time of 1min 30 s was employed. Cellular membranes can be identified throughout the gastrointestinal organs. Frequency domain image analysis can be used to distinguish columnar from squamous epithelium. Profilometric information on sample surfaces can be obtained directly as isophase lines. Second, the miniaturized confocal laser scanning microscope is characterized. Having an effective diameter of 25 mm, it houses single-mode optical fiber,scanning mirror and an objective lens. The micro-electro-mechanical mirror with gimballed suspension allows two dimensional scanning without introducing an optical path difference. The sinusoidal movement of both axes has to be considered to approximate cartesian image coordinates. Field geometry is illustrated s function of excitation amplitude and frequency. Acceptable image quality is chieved for frame rate of 0.5 Hz. A strategy to position the focal plane axially within the sample volume is discussed.

  7. Force feedback microscopy based on an optical beam deflection scheme

    NASA Astrophysics Data System (ADS)

    Vitorino, Miguel V.; Carpentier, Simon; Costa, Luca; Rodrigues, Mario S.

    2014-07-01

    Force feedback microscopy circumvents the jump to contact in atomic force microscopy when using soft cantilevers and quantitatively measures the interaction properties at the nanoscale by simultaneously providing force, force gradient, and dissipation. The force feedback microscope developed so far used an optical cavity to measure the tip displacement. In this Letter, we show that the more conventional optical beam deflection scheme can be used to the same purpose. With this instrument, we have followed the evolution of the Brownian motion of the tip under the influence of a water bridge.

  8. Force feedback microscopy based on an optical beam deflection scheme

    SciTech Connect

    Vitorino, Miguel V.; Rodrigues, Mario S.; Carpentier, Simon; Costa, Luca

    2014-07-07

    Force feedback microscopy circumvents the jump to contact in atomic force microscopy when using soft cantilevers and quantitatively measures the interaction properties at the nanoscale by simultaneously providing force, force gradient, and dissipation. The force feedback microscope developed so far used an optical cavity to measure the tip displacement. In this Letter, we show that the more conventional optical beam deflection scheme can be used to the same purpose. With this instrument, we have followed the evolution of the Brownian motion of the tip under the influence of a water bridge.

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

    PubMed

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

    2012-12-18

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

  10. Probing graphene defects and estimating graphene quality with optical microscopy

    SciTech Connect

    Lai, Shen; Kyu Jang, Sung; Jae Song, Young; Lee, Sungjoo

    2014-01-27

    We report a simple and accurate method for detecting graphene defects that utilizes the mild, dry annealing of graphene/Cu films in air. In contrast to previously reported techniques, our simple approach with optical microscopy can determine the density and degree of dislocation of defects in a graphene film without inducing water-related damage or functionalization. Scanning electron microscopy, confocal Raman and atomic force microscopy, and X-ray photoelectron spectroscopy analysis were performed to demonstrate that our nondestructive approach to characterizing graphene defects with optimized thermal annealing provides rapid and comprehensive determinations of graphene quality.

  11. Where Do We Stand with Super-Resolution Optical Microscopy?

    PubMed

    Nienhaus, Karin; Nienhaus, G Ulrich

    2016-01-29

    Super-resolution fluorescence microscopy has become an invaluable, powerful approach to study biomolecular dynamics and interactions via selective labeling and observation of specific molecules in living cells, tissues and even entire organisms. In this perspective, we present a brief overview of the main techniques and their application to cellular biophysics. We place special emphasis on super-resolution imaging via single-molecule localization microscopy and stimulated emission depletion/reversible saturable optical fluorescence transitions microscopy, and we also briefly address fluorescence fluctuation approaches, notably raster image correlation spectroscopy, as tools to record fast diffusion and transport. PMID:26743847

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

    NASA Astrophysics Data System (ADS)

    U-Thainual, Paweena; Kim, Do-Hyun

    2015-12-01

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

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

    PubMed

    U-Thainual, Paweena; Kim, Do-Hyun

    2015-12-01

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

  14. Simulations of atomic resolution tip-enhanced optical microscopy

    NASA Astrophysics Data System (ADS)

    Downes, Andrew; Salter, Donald; Elfick, Alistair

    2006-11-01

    Optical techniques can access a wealth of information but traditionally their resolution has been restricted by the diffraction limit. Near-field techniques, which used nanoscale apertures or nanotip electric field enhancement, have succeeded in circumventing Abbe’s law. We show that atomic resolution is theoretically achievable for tip enhanced optical microscopy. Using finite element analysis of the electromagnetic field around a small radius metallic scanning probe microscopy tip, we modeled various tip radii and materials, and an aqueous environment as well as ambient air. For a 1 nm gold tip we predict a strong red shift, and surprisingly high values for the enhancement of the intensity of scattered light over 107. For this tip, we predict that 0.2 nm lateral resolution in optical imaging is achievable good enough to resolve individual atomic bonds. The promise of optical data at these spatial scales offers great potential for nanometrology and nanotechnology applications.

  15. Optical characters of prostate using nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Wu, Shulian; Li, Hui; Zhang, Xiaoman; Wang, Yunxia; Peng, Dongqing

    2012-12-01

    The incidence rate of the prostatic hyperplasia is increasing in near decade, early detection is important for preventing the prostatic cancer (PCa). In this study, the images of prostate and cavernous nerves were carried out using intrinsic fluorescence and scattering properties of the tissues without any exogenous dye or contrast agent based on nonlinear optical microscope. The texture feature and optical property of the interfibrillar substance in prostate tissue were extracted and analyzed for charactering the prostate structure. It will be the feature parameter to differentiate the normal, the inflammation or cancer of prostate tissue in clinical with the application of miniature endoscope nonlinear optical microscope in vivo.

  16. Incorporating Basic Optical Microscopy in the Instrumental Analysis Laboratory

    ERIC Educational Resources Information Center

    Flowers, Paul A.

    2011-01-01

    A simple and versatile approach to incorporating basic optical microscopy in the undergraduate instrumental analysis laboratory is described. Attaching a miniature CCD spectrometer to the video port of a standard compound microscope yields a visible microspectrophotometer suitable for student investigations of fundamental spectrometry concepts,…

  17. Laser-scanning optical-resolution photoacoustic microscopy.

    PubMed

    Xie, Zhixing; Jiao, Shuliang; Zhang, Hao F; Puliafito, Carmen A

    2009-06-15

    We have developed a laser-scanning optical-resolution photoacoustic microscopy method that can potentially fuse with existing optical microscopic imaging modalities. To acquire an image, the ultrasonic transducer is kept stationary during data acquisition, and only the laser light is raster scanned by an x-y galvanometer scanner. A lateral resolution of 7.8 microm and a circular field of view with a diameter of 6 mm were achieved in an optically clear medium. Using a laser system working at a pulse repetition rate of 1,024 Hz, the data acquisition time for an image consisting of 256 x 256 pixels was less than 2 min. PMID:19529698

  18. Atomic force microscopy combined with optical tweezers (AFM/OT)

    NASA Astrophysics Data System (ADS)

    Pierini, F.; Zembrzycki, K.; Nakielski, P.; Pawłowska, S.; Kowalewski, T. A.

    2016-02-01

    The role of mechanical properties is essential to understand molecular, biological materials, and nanostructures dynamics and interaction processes. Atomic force microscopy (AFM) is the most commonly used method of direct force evaluation, but due to its technical limitations this single probe technique is unable to detect forces with femtonewton resolution. In this paper we present the development of a combined atomic force microscopy and optical tweezers (AFM/OT) instrument. The focused laser beam, on which optical tweezers are based, provides us with the ability to manipulate small dielectric objects and to use it as a high spatial and temporal resolution displacement and force sensor in the same AFM scanning zone. We demonstrate the possibility to develop a combined instrument with high potential in nanomechanics, molecules manipulation and biological studies. AFM/OT equipment is described and characterized by studying the ability to trap dielectric objects and quantifying the detectable and applicable forces. Finally, optical tweezers calibration methods and instrument applications are given.

  19. In vivo switchable optical- and acoustic-resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Jeon, Seungwan; Kim, Jaewoo; Kim, Chulhong

    2016-03-01

    Photoacoustic microscopy (PAM) provides high resolution and large penetration depth by utilizing the high optical sensitivity and low scattering of ultrasound. Hybrid PAM systems can be classified into two categories: opticalresolution photoacoustic microscopy (OR-PAM) and acoustic-resolution photoacoustic microscopy (AR-PAM). ORPAM provides a very high lateral resolution with a strong optical focus, but the penetration depth is limited to one optical transport mean free path. AR-PAM provides a relatively greater penetration depth using diffused light in biological tissues. The resolution of AR-PAM is determined by its ultrasonic parameters. In this study, we performed an in vivo testing of a switchable OR-/AR-PAM system. In this system, two modes can be switched by changing its collimator lens and optical fiber. The lateral resolution of OR-PAM was measured using a resolution test target, and the full width at half maximum (FWHM) of the edge spread function was 2.5 μm. To calculate the lateral resolution of ARPAM, a 6-μm-diameter carbon fiber was used, and the FWHM of the line spread function was 80.2 μm. We successfully demonstrated the multiscale imaging capability of the switchable OR-/AR-PAM system by visualizing microvascular networks in mouse ears, brain, legs, skin, and eyes.

  20. Sensorless adaptive optics implementation in widefield optical sectioning microscopy inside in vivo Drosophila brain

    NASA Astrophysics Data System (ADS)

    Pedrazzani, Mélanie; Loriette, Vincent; Tchenio, Paul; Benrezzak, Sakina; Nutarelli, Daniele; Fragola, Alexandra

    2016-03-01

    We present an implementation of a sensorless adaptive optics loop in a widefield fluorescence microscope. This setup is designed to compensate for aberrations induced by the sample on both excitation and emission pathways. It allows fast optical sectioning inside a living Drosophila brain. We present a detailed characterization of the system performances. We prove that the gain brought to optical sectioning by realizing structured illumination microscopy with adaptive optics down to 50 μm deep inside living Drosophila brain.

  1. Sensorless adaptive optics implementation in widefield optical sectioning microscopy inside in vivo Drosophila brain.

    PubMed

    Pedrazzani, Mélanie; Loriette, Vincent; Tchenio, Paul; Benrezzak, Sakina; Nutarelli, Daniele; Fragola, Alexandra

    2016-03-01

    We present an implementation of a sensorless adaptive optics loop in a widefield fluorescence microscope. This setup is designed to compensate for aberrations induced by the sample on both excitation and emission pathways. It allows fast optical sectioning inside a living Drosophila brain. We present a detailed characterization of the system performances. We prove that the gain brought to optical sectioning by realizing structured illumination microscopy with adaptive optics down to 50 μm deep inside living Drosophila brain. PMID:26968001

  2. Adaptive optics two-photon scanning laser fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Zhou, Yaopeng; Bifano, Thomas; Lin, Charles

    2011-03-01

    Two-photon fluorescence microscopy provides a powerful tool for deep tissue imaging. However, optical aberrations from illumination beam path limit imaging depth and resolution. Adaptive Optics (AO) is found to be useful to compensate for optical aberrations and improve image resolution and contrast from two-photon excitation. We have developed an AO system relying on a MEMS Deformable Mirror (DM) to compensate the optical aberrations in a two-photon scanning laser fluorescence microscope. The AO system utilized a Zernike polynomial based stochastic parallel gradient descent (SPGD) algorithm to optimize the DM shape for wavefront correction. The developed microscope is applied for subsurface imaging of mouse bone marrow. It was demonstrated that AO allows 80% increase in fluorescence signal intensity from bone cavities 145um below the surface. The AO-enhanced microscope provides cellular level images of mouse bone marrow at depths exceeding those achievable without AO.

  3. Mosaic acquisition and processing for optical-resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Shao, Peng; Shi, Wei; Chee, Ryan K. W.; Zemp, Roger J.

    2012-08-01

    In optical-resolution photo-acoustic microscopy (OR-PAM), data acquisition time is limited by both laser pulse repetition rate (PRR) and scanning speed. Optical-scanning offers high speed, but limited, field of view determined by ultrasound transducer sensitivity. In this paper, we propose a hybrid optical and mechanical-scanning OR-PAM system with mosaic data acquisition and processing. The system employs fast-scanning mirrors and a diode-pumped, nanosecond-pulsed, Ytterbium-doped, 532-nm fiber laser with PRR up to 600 kHz. Data from a sequence of image mosaic patches is acquired systematically, at predetermined mechanical scanning locations, with optical scanning. After all imaging locations are covered, a large panoramic scene is generated by stitching the mosaic patches together. Our proposed system is proven to be at least 20 times faster than previous reported OR-PAM systems.

  4. Holographic fluorescence microscopy with incoherent digital holographic adaptive optics.

    PubMed

    Jang, Changwon; Kim, Jonghyun; Clark, David C; Lee, Seungjae; Lee, Byoungho; Kim, Myung K

    2015-01-01

    Introduction of adaptive optics technology into astronomy and ophthalmology has made great contributions in these fields, allowing one to recover images blurred by atmospheric turbulence or aberrations of the eye. Similar adaptive optics improvement in microscopic imaging is also of interest to researchers using various techniques. Current technology of adaptive optics typically contains three key elements: a wavefront sensor, wavefront corrector, and controller. These hardware elements tend to be bulky, expensive, and limited in resolution, involving, for example, lenslet arrays for sensing or multiactuator deformable mirrors for correcting. We have previously introduced an alternate approach based on unique capabilities of digital holography, namely direct access to the phase profile of an optical field and the ability to numerically manipulate the phase profile. We have also demonstrated that direct access and compensation of the phase profile are possible not only with conventional coherent digital holography, but also with a new type of digital holography using incoherent light: selfinterference incoherent digital holography (SIDH). The SIDH generates a complex—i.e., amplitude plus phase—hologram from one or several interferograms acquired with incoherent light, such as LEDs, lamps, sunlight, or fluorescence. The complex point spread function can be measured using guide star illumination and it allows deterministic deconvolution of the full-field image. We present experimental demonstration of aberration compensation in holographic fluorescence microscopy using SIDH. Adaptive optics by SIDH provides new tools for improved cellular fluorescence microscopy through intact tissue layers or other types of aberrant media. PMID:26146767

  5. Holographic fluorescence microscopy with incoherent digital holographic adaptive optics

    NASA Astrophysics Data System (ADS)

    Jang, Changwon; Kim, Jonghyun; Clark, David C.; Lee, Seungjae; Lee, Byoungho; Kim, Myung K.

    2015-11-01

    Introduction of adaptive optics technology into astronomy and ophthalmology has made great contributions in these fields, allowing one to recover images blurred by atmospheric turbulence or aberrations of the eye. Similar adaptive optics improvement in microscopic imaging is also of interest to researchers using various techniques. Current technology of adaptive optics typically contains three key elements: a wavefront sensor, wavefront corrector, and controller. These hardware elements tend to be bulky, expensive, and limited in resolution, involving, for example, lenslet arrays for sensing or multiactuator deformable mirrors for correcting. We have previously introduced an alternate approach based on unique capabilities of digital holography, namely direct access to the phase profile of an optical field and the ability to numerically manipulate the phase profile. We have also demonstrated that direct access and compensation of the phase profile are possible not only with conventional coherent digital holography, but also with a new type of digital holography using incoherent light: self­interference incoherent digital holography (SIDH). The SIDH generates a complex-i.e., amplitude plus phase-hologram from one or several interferograms acquired with incoherent light, such as LEDs, lamps, sunlight, or fluorescence. The complex point spread function can be measured using guide star illumination and it allows deterministic deconvolution of the full-field image. We present experimental demonstration of aberration compensation in holographic fluorescence microscopy using SIDH. Adaptive optics by SIDH provides new tools for improved cellular fluorescence microscopy through intact tissue layers or other types of aberrant media.

  6. Tip-enhanced near-field optical microscopy

    PubMed Central

    Mauser, Nina; Hartschuh, Achim

    2013-01-01

    Tip-enhanced near-field optical microscopy (TENOM) is a scanning probe technique capable of providing a broad range of spectroscopic information on single objects and structured surfaces at nanometer spatial resolution and with highest detection sensitivity. In this review, we first illustrate the physical principle of TENOM that utilizes the antenna function of a sharp probe to efficiently couple light to excitations on nanometer length scales. We then discuss the antenna-induced enhancement of different optical sample responses including Raman scattering, fluorescence, generation of photocurrent and electroluminescence. Different experimental realizations are presented and several recent examples that demonstrate the capabilities of the technique are reviewed. PMID:24100541

  7. The Forces at Play in Optical Force Microscopy

    NASA Astrophysics Data System (ADS)

    Brocious, Jordan

    Optical force microscopy is a novel technique where mechanical detection with a cantilevered probe replaces the detection of photons to investigate optically induced processes and states. A theoretical and experimental analysis is performed here of the forces present in optical force microscopy operated in tapping mode, which reveals two dominant optically induced forces, the gradient force and the scattering force. Force-distance curves are reconstructed from experimental amplitude and phase information for glass, gold nanowires and molecular clusters of silicon naphtalocyanine samples. The scattering force is shown to be insensitive to both nano-scale tip-sample distances and sample polarizability and is dependent on the form of the tip. The gradient force demonstrates a z-4 tip-sample distance dependence, localized to a few nanometers, and is strongly dependent on the polarizability of the sample which enables spectroscopic imaging through force detection. The different distance-dependence and polarizability-dependence of the gradient and scattering forces give rise to a complex force-distance curve which determines imaging contrast along with the cantilever set-point, knowledge of which is essential for image interpretation.

  8. Parameter optimization for through-focus scanning optical microscopy.

    PubMed

    Attota, Ravi Kiran; Kang, Hyeonggon

    2016-06-27

    It is important to economically and non-destructively analyze three-dimensional (3-D) shapes of nanometer to micrometer scale objects with sub-nanometer measurement resolution for emerging high-volume nanomanufacturing, especially for process control. High-throughput through-focus scanning optical microscopy (TSOM) demonstrates promise for such applications. TSOM uses a conventional optical microscope for 3-D shape metrology by making use of the complete set of through-focus, four-dimensional optical data. However, a systematic study showing the effect of various parameters on the TSOM method is lacking. Here we present the optimization of the basic parameters such as illumination numerical aperture (NA), collection NA, focus step height, digital camera pixel size, illumination polarization, and illumination wavelength to achieve peak performance of the TSOM method. PMID:27410642

  9. Polarization sensitive optical coherence microscopy for brain imaging.

    PubMed

    Wang, Hui; Akkin, Taner; Magnain, Caroline; Wang, Ruopeng; Dubb, Jay; Kostis, William J; Yaseen, Mohammad A; Cramer, Avilash; Sakadžić, Sava; Boas, David

    2016-05-15

    Optical coherence tomography (OCT) and optical coherence microscopy (OCM) have demonstrated the ability to investigate cyto- and myelo-architecture in the brain. Polarization-sensitive OCT provides sensitivity to additional contrast mechanisms, specifically the birefringence of myelination and, therefore, is advantageous for investigating white matter fiber tracts. In this Letter, we developed a polarization-sensitive optical coherence microscope (PS-OCM) with a 3.5 μm axial and 1.3 μm transverse resolution to investigate fiber organization and orientation at a finer scale than previously demonstrated with PS-OCT. In a reconstructed mouse brain section, we showed that at the focal depths of 20-70 μm, the PS-OCM reliably identifies the neuronal fibers and quantifies the in-plane orientation. PMID:27176965

  10. Multifocal optical-resolution photoacoustic microscopy in reflection mode

    NASA Astrophysics Data System (ADS)

    Li, Guo; Maslov, Konstantin I.; Wang, Lihong V.

    2013-03-01

    Compared with single-focus optical-resolution photoacoustic microscopy (OR-PAM), multifocal OR-PAM utilizes both multifocal optical illumination and an ultrasonic array transducer, significantly increasing the imaging speed. Here we present a reflection-mode multifocal OR-PAM system based on a microlens array that provides multiple foci and an ultrasonic array transducer that receives the excited photoacoustic waves from all foci simultaneously. By using a customized microprism to reflect the incident laser beam to the microlens array, we align the multiple optical foci confocally with the focal zone of the ultrasonic array transducer. Experiments show our reflection-mode multifocal ORPAM system is capable of imaging microvessels in vivo, and it can image a 9 mm x 5 mm x 2.5 mm volume at 16 μm lateral resolution in ~4 min, limited by the signal multiplexing ratio and laser pulse repetition rate.

  11. Reflection-mode multifocal optical-resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Li, Guo; Maslov, Konstantin I.; Wang, Lihong V.

    2013-03-01

    Compared with single-focus optical-resolution photoacoustic microscopy (OR-PAM), multifocal OR-PAM utilizes both multifocal optical illumination and an ultrasonic array transducer, significantly increasing the imaging speed. A reflection-mode multifocal OR-PAM system based on a microlens array that provides multiple foci as well as an ultrasonic array transducer that receives the excited photoacoustic waves from all foci simultaneously is presented. Using a customized microprism to reflect the incident laser beam to the microlens array, the multiple optical foci are aligned confocally with the focal zone of the ultrasonic array transducer. Experiments show the reflection-mode multifocal OR-PAM is capable of imaging microvessels in vivo, and it can image a 6×5×2.5 mm3 volume at 16 μm lateral resolution in ˜2.5 min, which was limited by the signal multiplexing ratio and laser pulse repetition rate.

  12. Holographic fluorescence microscopy with incoherent digital holographic adaptive optics

    NASA Astrophysics Data System (ADS)

    Jang, Changwon; Kim, Jonghyun; Clark, David C.; Lee, Byoungho; Kim, Myung K.

    2015-03-01

    Introduction of adaptive optics technology into astronomy and ophthalmology has made great contributions in these fields, allowing one to recover images blurred by atmospheric turbulence or aberrations of the eye. Similar adaptive optics improvement in microscopic imaging is also of interest to researchers using various techniques. Current technology of adaptive optics typically contains three key elements: wavefront sensor, wavefront corrector and controller. These hardware elements tend to be bulky, expensive, and limited in resolution, involving, e.g., lenslet arrays for sensing or multi-acuator deformable mirrors for correcting. We have previously introduced an alternate approach to adaptive optics based on unique capabilities of digital holography, namely direct access to the phase profile of an optical field and the ability to numerically manipulate the phase profile. We have also demonstrated that direct access and compensation of the phase profile is possible not only with the conventional coherent type of digital holography, but also with a new type of digital holography using incoherent light: self-interference incoherent digital holography (SIDH). The SIDH generates complex - i.e. amplitude plus phase - hologram from one or several interferograms acquired with incoherent light, such as LEDs, lamps, sunlight, or fluorescence. The complex point spread function can be measured using a guide star illumination and it allows deterministic deconvolution of the full-field image. We present experimental demonstration of aberration compensation in holographic fluorescence microscopy using SIDH. The adaptive optics by SIDH provides new tools for improved cellular fluorescence microscopy through intact tissue layers or other types of aberrant media.

  13. Correlative super-resolution fluorescence microscopy combined with optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Kim, Sungho; Kim, Gyeong Tae; Jang, Soohyun; Shim, Sang-Hee; Bae, Sung Chul

    2015-03-01

    Recent development of super-resolution fluorescence imaging technique such as stochastic optical reconstruction microscopy (STORM) and photoactived localization microscope (PALM) has brought us beyond the diffraction limits. It allows numerous opportunities in biology because vast amount of formerly obscured molecular structures, due to lack of spatial resolution, now can be directly observed. A drawback of fluorescence imaging, however, is that it lacks complete structural information. For this reason, we have developed a super-resolution multimodal imaging system based on STORM and full-field optical coherence microscopy (FF-OCM). FF-OCM is a type of interferometry systems based on a broadband light source and a bulk Michelson interferometer, which provides label-free and non-invasive visualization of biological samples. The integration between the two systems is simple because both systems use a wide-field illumination scheme and a conventional microscope. This combined imaging system gives us both functional information at a molecular level (~20nm) and structural information at the sub-cellular level (~1μm). For thick samples such as tissue slices, while FF-OCM is readily capable of imaging the 3D architecture, STORM suffer from aberrations and high background fluorescence that substantially degrade the resolution. In order to correct the aberrations in thick tissues, we employed an adaptive optics system in the detection path of the STORM microscope. We used our multimodal system to obtain images on brain tissue samples with structural and functional information.

  14. Automated seeding-based nuclei segmentation in nonlinear optical microscopy.

    PubMed

    Medyukhina, Anna; Meyer, Tobias; Heuke, Sandro; Vogler, Nadine; Dietzek, Benjamin; Popp, Jürgen

    2013-10-01

    Nonlinear optical (NLO) microscopy based, e.g., on coherent anti-Stokes Raman scattering (CARS) or two-photon-excited fluorescence (TPEF) is a fast label-free imaging technique, with a great potential for biomedical applications. However, NLO microscopy as a diagnostic tool is still in its infancy; there is a lack of robust and durable nuclei segmentation methods capable of accurate image processing in cases of variable image contrast, nuclear density, and type of investigated tissue. Nonetheless, such algorithms specifically adapted to NLO microscopy present one prerequisite for the technology to be routinely used, e.g., in pathology or intraoperatively for surgical guidance. In this paper, we compare the applicability of different seeding and boundary detection methods to NLO microscopic images in order to develop an optimal seeding-based approach capable of accurate segmentation of both TPEF and CARS images. Among different methods, the Laplacian of Gaussian filter showed the best accuracy for the seeding of the image, while a modified seeded watershed segmentation was the most accurate in the task of boundary detection. The resulting combination of these methods followed by the verification of the detected nuclei performs high average sensitivity and specificity when applied to various types of NLO microscopy images. PMID:24085213

  15. Super-resolution microscopy of single atoms in optical lattices

    NASA Astrophysics Data System (ADS)

    Alberti, Andrea; Robens, Carsten; Alt, Wolfgang; Brakhane, Stefan; Karski, Michał; Reimann, René; Widera, Artur; Meschede, Dieter

    2016-05-01

    We report on image processing techniques and experimental procedures to determine the lattice-site positions of single atoms in an optical lattice with high reliability, even for limited acquisition time or optical resolution. Determining the positions of atoms beyond the diffraction limit relies on parametric deconvolution in close analogy to methods employed in super-resolution microscopy. We develop a deconvolution method that makes effective use of the prior knowledge of the optical transfer function, noise properties, and discreteness of the optical lattice. We show that accurate knowledge of the image formation process enables a dramatic improvement on the localization reliability. This allows us to demonstrate super-resolution of the atoms’ position in closely packed ensembles where the separation between particles cannot be directly optically resolved. Furthermore, we demonstrate experimental methods to precisely reconstruct the point spread function with sub-pixel resolution from fluorescence images of single atoms, and we give a mathematical foundation thereof. We also discuss discretized image sampling in pixel detectors and provide a quantitative model of noise sources in electron multiplying CCD cameras. The techniques developed here are not only beneficial to neutral atom experiments, but could also be employed to improve the localization precision of trapped ions for ultra precise force sensing.

  16. Assessment of fibrotic liver disease with multimodal nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Lu, Fake; Zheng, Wei; Tai, Dean C. S.; Lin, Jian; Yu, Hanry; Huang, Zhiwei

    2010-02-01

    Liver fibrosis is the excessive accumulation of extracellular matrix proteins such as collagens, which may result in cirrhosis, liver failure, and portal hypertension. In this study, we apply a multimodal nonlinear optical microscopy platform developed to investigate the fibrotic liver diseases in rat models established by performing bile duct ligation (BDL) surgery. The three nonlinear microscopy imaging modalities are implemented on the same sectioned tissues of diseased model sequentially: i.e., second harmonic generation (SHG) imaging quantifies the contents of the collagens, the two-photon excitation fluorescence (TPEF) imaging reveals the morphology of hepatic cells, while coherent anti-Stokes Raman scattering (CARS) imaging maps the distributions of fats or lipids quantitatively across the tissue. Our imaging results show that during the development of liver fibrosis (collagens) in BDL model, fatty liver disease also occurs. The aggregated concentrations of collagen and fat constituents in liver fibrosis model show a certain correlationship between each other.

  17. Quantitative interferometric microscopy cytometer based on regularized optical flow algorithm

    NASA Astrophysics Data System (ADS)

    Xue, Liang; Vargas, Javier; Wang, Shouyu; Li, Zhenhua; Liu, Fei

    2015-09-01

    Cell detections and analysis are important in various fields, such as medical observations and disease diagnoses. In order to analyze the cell parameters as well as observe the samples directly, in this paper, we present an improved quantitative interferometric microscopy cytometer, which can monitor the quantitative phase distributions of bio-samples and realize cellular parameter statistics. The proposed system is able to recover the phase imaging of biological samples in the expanded field of view via a regularized optical flow demodulation algorithm. This algorithm reconstructs the phase distribution with high accuracy with only two interferograms acquired at different time points simplifying the scanning system. Additionally, the method is totally automatic, and therefore it is convenient for establishing a quantitative phase cytometer. Moreover, the phase retrieval approach is robust against noise and background. Excitingly, red blood cells are readily investigated with the quantitative interferometric microscopy cytometer system.

  18. Nonlinear Optical Microscopy Signal Processing Strategies in Cancer

    PubMed Central

    Adur, Javier; Carvalho, Hernandes F; Cesar, Carlos L; Casco, Víctor H

    2014-01-01

    This work reviews the most relevant present-day processing methods used to improve the accuracy of multimodal nonlinear images in the detection of epithelial cancer and the supporting stroma. Special emphasis has been placed on methods of non linear optical (NLO) microscopy image processing such as: second harmonic to autofluorescence ageing index of dermis (SAAID), tumor-associated collagen signatures (TACS), fast Fourier transform (FFT) analysis, and gray level co-occurrence matrix (GLCM)-based methods. These strategies are presented as a set of potential valuable diagnostic tools for early cancer detection. It may be proposed that the combination of NLO microscopy and informatics based image analysis approaches described in this review (all carried out on free software) may represent a powerful tool to investigate collagen organization and remodeling of extracellular matrix in carcinogenesis processes. PMID:24737930

  19. Nonlinear optical microscopy improvement by focal-point axial modulation

    NASA Astrophysics Data System (ADS)

    Dashtabi, Mahdi Mozdoor; Massudi, Reza

    2016-05-01

    Among the most important challenges of microscopy-even more important than the resolution enhancement, especially in biological and neuroscience applications-is noninvasive and label-free imaging deeper into live scattering samples. However, the fundamental limitation on imaging depth is the signal-to-background ratio in scattering biological tissues. Here, using a vibrating microscope objective in conjunction with a lock-in amplifier, we demonstrate the background cancellation in imaging the samples surrounded by turbid and scattering media, which leads to more clear images deeper into the samples. Furthermore, this technique offers the localization and resolution enhancement as well as resolves ambiguities in signal interpretation, using a single-color laser. This technique is applicable to most nonlinear as well as some linear point-scanning optical microscopies.

  20. Vertically integrated optics for ballistic electron emission luminescence microscopy

    NASA Astrophysics Data System (ADS)

    Appelbaum, Ian; Yi, Wei; Russell, K. J.; Narayanamurti, V.; Hanson, M. P.; Gossard, A. C.

    2005-02-01

    We have integrated a photon detector directly into a ballistic electron emission luminescence (BEEL) heterostructure, just below a luminescent quantum well. Results from solid-state metal-base hot-electron transistors fabricated with this collector design indicate that more than 10% of the photons emitted by the quantum well excite photoelectrons in the detector region. The improved photonic coupling and effective collection angle in this scheme improves the BEEL signal by many orders of magnitude as compared to far-field detection with the most sensitive single-photon counters, enabling BEEL microscopy in systems with no optical components.

  1. Ultrafast Optical Microscopy of Single Monolayer Molybdenum Disulfide Flakes

    PubMed Central

    Seo, Minah; Yamaguchi, Hisato; Mohite, Aditya D.; Boubanga-Tombet, Stephane; Blancon, Jean-Christophe; Najmaei, Sina; Ajayan, Pulickel M.; Lou, Jun; Taylor, Antoinette J.; Prasankumar, Rohit P.

    2016-01-01

    We have performed ultrafast optical microscopy on single flakes of atomically thin CVD-grown molybdenum disulfide, using non-degenerate femtosecond pump-probe spectroscopy to excite and probe carriers above and below the indirect and direct band gaps. These measurements reveal the influence of layer thickness on carrier dynamics when probing near the band gap. Furthermore, fluence-dependent measurements indicate that carrier relaxation is primarily influenced by surface-related defect and trap states after above-bandgap photoexcitation. The ability to probe femtosecond carrier dynamics in individual flakes can thus give much insight into light-matter interactions in these two-dimensional nanosystems. PMID:26876194

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

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

  3. Dark-field circular depolarization optical coherence microscopy

    PubMed Central

    Mehta, Kalpesh; Zhang, Pengfei; Yeo, Eugenia Li Ling; Kah, James Chen Yong; Chen, Nanguang

    2013-01-01

    Optical coherence microscopy (OCM) is a widely used structural imaging modality. To extend its application in molecular imaging, gold nanorods are widely used as contrast agents for OCM. However, they very often offer limited sensitivity as a result of poor signal to background ratio. Here we experimentally demonstrate that a novel OCM implementation based on dark-field circular depolarization detection can efficiently detect circularly depolarized signal from gold nanorods and at the same time efficiently suppress the background signals. This results into a significant improvement in signal to background ratio. PMID:24049689

  4. Combined two-photon microscopy and angiographic optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Kim, Bumju; Wang, Tae Jun; Li, Qingyun; Nam, Jutaek; Hwang, Sekyu; Chung, Euiheon; Kim, Sungjee; Kim, Ki Hean

    2013-08-01

    A combined two-photon microscopy (TPM) and angiographic optical coherence tomography (OCT) is developed, which can provide molecular, cellular, structural, and vascular information of tissue specimens in vivo. This combined system is implemented by adding an OCT vasculature visualization method to the previous combined TPM and OCT, and then is applied to in vivo tissue imaging. Two animal models, a mouse brain cranial window model and a mouse ear cancer model, are used. Both molecular, cellular information at local regions of tissues, and structural, vascular information at relatively larger regions are visualized in the same sections. In vivo tissue microenvironments are better elucidated by the combined TPM and angiographic OCT.

  5. Ultrafast Optical Microscopy of Single Monolayer Molybdenum Disulfide Flakes

    DOE PAGESBeta

    Seo, Minah; Yamaguchi, Hisato; Mohite, Aditya D.; Boubanga-Tombet, Stephane; Blancon, Jean-Christophe; Najmaei, Sina; Ajayan, Pulickel M.; Lou, Jun; Taylor, Antoinette J.; Prasankumar, Rohit P.

    2016-02-15

    We performed ultrafast optical microscopy on single flakes of atomically thin CVD-grown molybdenum disulfide, using non-degenerate femtosecond pump-probe spectroscopy to excite and probe carriers above and below the indirect and direct band gaps. These measurements reveal the influence of layer thickness on carrier dynamics when probing near the band gap. Furthermore, fluence-dependent measurements indicate that carrier relaxation is primarily influenced by surface-related defect and trap states after above-bandgap photoexcitation. Furthermore, the ability to probe femtosecond carrier dynamics in individual flakes can thus give much insight into light-matter interactions in these two-dimensional nanosystems.

  6. Near-field optical microscopy based on microfabricated probes.

    PubMed

    Eckert, R; Freyland, J M; Gersen, H; Heinzelmann, H; Schürmann, G; Noell, W; Staufer, U; De Rooij, N F

    2001-04-01

    We demonstrate high resolution imaging with microfabricated, cantilevered probes, consisting of solid quartz tips on silicon levers. The tips are covered by a 60-nm thick layer of aluminium, which appears to be closed at the apex when investigated by transmission electron microscopy. An instrument specifically built for cantilever probes was used to record images of latex bead projection patterns in transmission as well as single molecule fluorescence. All images were recorded in constant height mode and show optical resolutions down to 32 nm. PMID:11298861

  7. Ultrafast Optical Microscopy of Single Monolayer Molybdenum Disulfide Flakes.

    PubMed

    Seo, Minah; Yamaguchi, Hisato; Mohite, Aditya D; Boubanga-Tombet, Stephane; Blancon, Jean-Christophe; Najmaei, Sina; Ajayan, Pulickel M; Lou, Jun; Taylor, Antoinette J; Prasankumar, Rohit P

    2016-01-01

    We have performed ultrafast optical microscopy on single flakes of atomically thin CVD-grown molybdenum disulfide, using non-degenerate femtosecond pump-probe spectroscopy to excite and probe carriers above and below the indirect and direct band gaps. These measurements reveal the influence of layer thickness on carrier dynamics when probing near the band gap. Furthermore, fluence-dependent measurements indicate that carrier relaxation is primarily influenced by surface-related defect and trap states after above-bandgap photoexcitation. The ability to probe femtosecond carrier dynamics in individual flakes can thus give much insight into light-matter interactions in these two-dimensional nanosystems. PMID:26876194

  8. Combined transmission and reflection optical microscopy of ice core sections

    NASA Astrophysics Data System (ADS)

    Binder, Tobias; Weikusat, Ilka; Kerst, Thomas; Eichler, Jan; Svensson, Anders; Bohleber, Pascal; Garbe, Christoph; Kipfstuhl, Sepp

    2013-04-01

    Microstructure analysis of ice cores is vital to understand the processes controlling the flow of ice on the microscale. To quantify the microstructural variability (and thus occurring processes) on centimeter, meter and kilometer scale along deep polar ice cores, a large number of sections has to be analyzed. In the last decade, two different methods have been applied: On the one hand, transmission optical microscopy of thin sections between crossed polarizers yields information on the distribution of crystal c-axes. On the other hand, reflection optical microscopy of polished and controlled sublimated section surfaces allows to characterize the high resolution properties of a single grain boundary, e.g. its length, shape or curvature. Based on a polar and an alpine ice core we applied both methods to the same set of sections. This enables us to combine all information on crystal orientation and (sub-)grain boundaries. In this contribution we introduce the method of combined transmission-polarization and reflection microscopy as well as an image processing framework for processing and matching both image types [1]. The information content of both analysis methods is limited and influenced by different types of artifacts. It is exemplary shown how the combination allows to compensate for deficiencies of one method. The gray values in images of the grain boundaries on polished ice core sections are influenced by the duration of surface sublimation and the energy/misorientation of the grain boundaries in the section. By combining these gray values with the misorientation obtained from the corresponding thin section imaged between crossed polarizers we try to validate the information content of gray values on the basis of large data sets. This approach is compared to X-ray Laue diffraction measurements (yielding full crystallographic orientation) which validated the sensitivity of the surface sublimation method [2]. As microscopy in transmission mode acquires volume

  9. Optical digital microscopy for cyto- and hematological studies in vitro

    NASA Astrophysics Data System (ADS)

    Ganilova, Yu. A.; Dolmashkin, A. A.; Doubrovski, V. A.; Yanina, I. Yu.; Tuchin, V. V.

    2013-08-01

    The dependence of the spatial resolution and field of view of an optical microscope equipped with a CCD camera on the objective magnification has been experimentally investigated. Measurement of these characteristics has shown that a spatial resolution of 20-25 px/μm at a field of view of about 110 μm is quite realistic; this resolution is acceptable for a detailed study of the processes occurring in cell. It is proposed to expand the dynamic range of digital camera by measuring and approximating its light characteristics with subsequent plotting of the corresponding calibration curve. The biological objects of study were human adipose tissue cells, as well as erythrocytes and their immune complexes in human blood; both objects have been investigated in vitro. Application of optical digital microscopy for solving specific problems of cytology and hematology can be useful in both biomedical studies in experiments with objects of nonbiological origin.

  10. Optimization-based wavefront sensorless adaptive optics for multiphoton microscopy.

    PubMed

    Antonello, Jacopo; van Werkhoven, Tim; Verhaegen, Michel; Truong, Hoa H; Keller, Christoph U; Gerritsen, Hans C

    2014-06-01

    Optical aberrations have detrimental effects in multiphoton microscopy. These effects can be curtailed by implementing model-based wavefront sensorless adaptive optics, which only requires the addition of a wavefront shaping device, such as a deformable mirror (DM) to an existing microscope. The aberration correction is achieved by maximizing a suitable image quality metric. We implement a model-based aberration correction algorithm in a second-harmonic microscope. The tip, tilt, and defocus aberrations are removed from the basis functions used for the control of the DM, as these aberrations induce distortions in the acquired images. We compute the parameters of a quadratic polynomial that is used to model the image quality metric directly from experimental input-output measurements. Finally, we apply the aberration correction by maximizing the image quality metric using the least-squares estimate of the unknown aberration. PMID:24977374

  11. Extending Single-Molecule Microscopy Using Optical Fourier Processing

    PubMed Central

    2015-01-01

    This article surveys the recent application of optical Fourier processing to the long-established but still expanding field of single-molecule imaging and microscopy. A variety of single-molecule studies can benefit from the additional image information that can be obtained by modulating the Fourier, or pupil, plane of a widefield microscope. After briefly reviewing several current applications, we present a comprehensive and computationally efficient theoretical model for simulating single-molecule fluorescence as it propagates through an imaging system. Furthermore, we describe how phase/amplitude-modulating optics inserted in the imaging pathway may be modeled, especially at the Fourier plane. Finally, we discuss selected recent applications of Fourier processing methods to measure the orientation, depth, and rotational mobility of single fluorescent molecules. PMID:24745862

  12. Laser Scanning-Assisted Tip-Enhanced Optical Microscopy for Robust Optical Nanospectroscopy.

    PubMed

    Yano, Taka-Aki; Tsuchimoto, Yuta; Mochizuki, Masahito; Hayashi, Tomohiro; Hara, Masahiko

    2016-07-01

    Laser-scanning-assisted tip-enhanced optical microscopy was developed for robust optical nanospectroscopy. The laser-scanning system was utilized to automatically set and keep the center of a tight laser-focusing spot in the proximity of a metallic tip with around 10 nm precision. This enabled us to efficiently and stably induce plasmon-coupled field enhancement at the apex of the metallic probe tip. The laser-scanning technique was also applied to tracking and compensating the thermal drift of the metallic tip in the spot. This technique is usable for long-term tip-enhanced optical spectroscopy without any optical degradation. PMID:27412187

  13. Endoscopic probe optics for spectrally encoded confocal microscopy

    PubMed Central

    Kang, DongKyun; Carruth, Robert W.; Kim, Minkyu; Schlachter, Simon C.; Shishkov, Milen; Woods, Kevin; Tabatabaei, Nima; Wu, Tao; Tearney, Guillermo J.

    2013-01-01

    Spectrally encoded confocal microscopy (SECM) is a form of reflectance confocal microscopy that can achieve high imaging speeds using relatively simple probe optics. Previously, the feasibility of conducting large-area SECM imaging of the esophagus in bench top setups has been demonstrated. Challenges remain, however, in translating SECM into a clinically-useable device; the tissue imaging performance should be improved, and the probe size needs to be significantly reduced so that it can fit into luminal organs of interest. In this paper, we report the development of new SECM endoscopic probe optics that addresses these challenges. A custom water-immersion aspheric singlet (NA = 0.5) was developed and used as the objective lens. The water-immersion condition was used to reduce the spherical aberrations and specular reflection from the tissue surface, which enables cellular imaging of the tissue deep below the surface. A custom collimation lens and a small-size grating were used along with the custom aspheric singlet to reduce the probe size. A dual-clad fiber was used to provide both the single- and multi- mode detection modes. The SECM probe optics was made to be 5.85 mm in diameter and 30 mm in length, which is small enough for safe and comfortable endoscopic imaging of the gastrointestinal tract. The lateral resolution was 1.8 and 2.3 µm for the single- and multi- mode detection modes, respectively, and the axial resolution 11 and 17 µm. SECM images of the swine esophageal tissue demonstrated the capability of this device to enable the visualization of characteristic cellular structural features, including basal cell nuclei and papillae, down to the imaging depth of 260 µm. These results suggest that the new SECM endoscopic probe optics will be useful for imaging large areas of the esophagus at the cellular scale in vivo. PMID:24156054

  14. Autofocus by Bayes Spectral Entropy Applied to Optical Microscopy.

    PubMed

    Podlech, Steffen

    2016-02-01

    This study introduces a passive autofocus method based on image analysis calculating the Bayes spectral entropy (BSE). The method is applied to optical microscopy and together with the specific construction of the opto-mechanical unit, it allows the analysis of large samples with complicated surfaces without subsampling. This paper will provide a short overview of the relevant theory of calculating the normalized discrete cosine transform when analyzing obtained images, in order to find the BSE measure. Furthermore, it will be shown that the BSE measure is a strong indicator, helping to determine the focal position of the optical microscope. To demonstrate the strength and robustness of the microscope system, tests have been performed using a 1951 USAF test pattern resolution chart determining the in focus position of the microscope. Finally, this method and the optical microscope system is applied to analyze an optical grating (100 lines/mm) demonstrating the detection of the focal position. The paper concludes with an outlook of potential applications of the presented system within quality control and surface analysis. PMID:26758956

  15. Multimodal nonlinear optical microscopy used to discriminate epithelial ovarian cancer

    NASA Astrophysics Data System (ADS)

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

    2011-07-01

    We used human specimens of epithelial ovarian cancer (serous type) to test the feasibility of nonlinear imaging as complementary tools for ovarian cancer diagnosis. Classical hematoxylin-and-eosin stained sections were applied to combining two-photon excitation fluorescence (TPEF), second (SHG), and third (THG) harmonic microscopy within the same imaging platform. We show that strong TPEF + SHG + THG signals can be obtained in fixed samples stained with Hematoxylin & Eosin (H&E) stored for a very long time and that H&E staining enhanced the THG signal. We demonstrate using anisotropy and morphological measurements, that SHG and THG of stained optical sections allow reproducible identification of neoplastic features such as architectural alterations of collagen fibrils at different stages of the neoplastic transformation and cellular atypia. Taken together, these results suggest that, with our viable imaging system, we can qualitatively and quantitatively assess endogenous optical biomarkers of the ovarian tissue with SHG and THG microscopy. This imaging capability may prove to be highly valuable in aiding to determine structural changes at the cellular and tissue levels, which may contribute to the development of new diagnostic techniques.

  16. A robotized six degree of freedom stage for optical microscopy

    NASA Astrophysics Data System (ADS)

    Avramov, M. Z.; Ivanov, I.; Pavlov, V.; Zaharieva, K.

    2013-04-01

    This work represents an investigation of the possibility to use a hexapod system for optical microscopy investigation and measurements. An appropriate hexapod stage has been developed. The stage has been calibrated and used for several different optical microscopy applications. The construction of the stage is based on the classic Stewart platform and thus represents a parallel robot with 6 degree of freedom. Appropriate software is controlling the transformation of the 3 position coordinates of the moving plate and the 3 Euler angles in position velocities and accelerations of the plate motion. An embedded microcontroller is implementing the motion plan and the PID controller regulating the kinematics. By difference to the available in the market hexapods the proposed solution is with lower precision but is significantly cheaper and simple to maintain. The repeatability obtained with current implementation is 0,05 mm and 0,001 rad. A specialized DSP based video processing engine is used for both feedback computation and application specific image processing in real-time. To verify the concept some applications has been developed for specific tasks and has been used for specific measurements.

  17. Modeling of optical quadrature microscopy for imaging mouse embryos

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

  18. Design of a handheld optical coherence microscopy endoscope

    NASA Astrophysics Data System (ADS)

    Korde, Vrushali R.; Liebmann, Erica; Barton, Jennifer K.

    2009-02-01

    Optical Coherence Microscopy (OCM) combines coherence gating, high numerical aperture optics, and a fiber core pinhole to provide high axial and lateral resolution with relatively large depth of imaging. We present a handheld rigid OCM endoscope with a 6 mm diameter tip, 1 mm scan width, and 1 mm imaging depth. This probe will allow noninvasive imaging of fine structural detail in vivo. X-Y scanning is performed distally with mirrors mounted to micro galvonometer scanners incorporated into the endoscope handle. Two scanning doublet lenses relay the stop from the galvonometers to the afocal relay stop. The endoscope optical design consists of an afocal Hopkins relay lens system and a 0.4 NA objective. To allow focusing at various depths in the tissue, the endoscope housing is designed in two pieces screwed together with a fine pitch threads. A small rotation of the outer housing moves the lenses proximal and distal relative to the window, causing the focal location in the tissue to change. The space between the final objective lens and the window is filled with distilled water to avoid misalignment of the focus and coherence gate. A knife edge test was performed and the line spread function FWHM was measured to be 2.25 μm. The MTF has at least 0.3 contrast at a 5 μm line pair. This rigid handheld OCM endoscope will be useful for application ranging from minimally invasive surgical imaging to assessing dysplasia and sun damage in skin.

  19. Exploring lipids with nonlinear optical microscopy in multiple biological systems

    NASA Astrophysics Data System (ADS)

    Alfonso-Garcia, Alba

    Lipids are crucial biomolecules for the well being of humans. Altered lipid metabolism may give rise to a variety of diseases that affect organs from the cardiovascular to the central nervous system. A deeper understanding of lipid metabolic processes would spur medical research towards developing precise diagnostic tools, treatment methods, and preventive strategies for reducing the impact of lipid diseases. Lipid visualization remains a complex task because of the perturbative effect exerted by traditional biochemical assays and most fluorescence markers. Coherent Raman scattering (CRS) microscopy enables interrogation of biological samples with minimum disturbance, and is particularly well suited for label-free visualization of lipids, providing chemical specificity without compromising on spatial resolution. Hyperspectral imaging yields large datasets that benefit from tailored multivariate analysis. In this thesis, CRS microscopy was combined with Raman spectroscopy and other label-free nonlinear optical techniques to analyze lipid metabolism in multiple biological systems. We used nonlinear Raman techniques to characterize Meibum secretions in the progression of dry eye disease, where the lipid and protein contributions change in ratio and phase segregation. We employed similar tools to examine lipid droplets in mice livers aboard a spaceflight mission, which lose their retinol content contributing to the onset of nonalcoholic fatty-liver disease. We also focused on atherosclerosis, a disease that revolves around lipid-rich plaques in arterial walls. We examined the lipid content of macrophages, whose variable phenotype gives rise to contrasting healing and inflammatory activities. We also proposed new label-free markers, based on lifetime imaging, for macrophage phenotype, and to detect products of lipid oxidation. Cholesterol was also detected in hepatitis C virus infected cells, and in specific strains of age-related macular degeneration diseased cells by

  20. Near-field scanning optical microscopy investigations of conjugated polymers

    NASA Astrophysics Data System (ADS)

    Dearo, Jessie Ann

    The Near-Field Scanning Optical Microscopy (NSOM) studies of novel, optically active, conjugated polymers are presented. NSOM is a relatively new technique which produces super resolution (˜50--100 nm) optical images simultaneously with topography. The conjugated polymer poly(p-phenylene vinylene) (PPV) and derivatives of PPV are organic semiconductor-like materials with interesting and unique optical properties. Derivatives of PPV have been used in LEDs and have potential in other optoelectronic devices. NSOM provides a tool for investigation of the photoluminescence, absorption/reflection, photo-dynamics and photoconductivity of films of PPV and PPV derivatives on the length scale that these properties are fundamentally defined. The NSOM experiments have revealed mesoscale domains (˜100 nm) of varying photoluminescence emission and average molecular order in drop cast films of PPV. NSOM of stretch-oriented PPV have shown domains of perpendicular molecular orientation with low photoluminescence emission. Near-field photoconductivity experiments of stretch-oriented PPV have correlated the mesoscale topography with the photoconductivity properties of the polymer. NSOM experiments of films of poly(2-methoxy, 5-(2'-(ethyl(hexyloxy)-p-phenylene vinylene) (MEH-PPV) have shown that there is mesoscale spatial inhomogeneity in the photo-oxidation process which reduces photoluminescence emission. NSOM has also been used to create nanoscale photo-patterning in MEH-PPV films. The NSOM experiments of blended films of MEH-PPV in polystyrene have shown mesoscale phase separation directly correlated to variations in the optical properties of the film. Derivatives of PPV, stretch-oriented in polyethylene, show photoluminescence intensity variations perpendicular and parallel to the stretch-direction correlated to topography features. As a complement to the NSOM studies of conjugated polymers, single polymer molecule experiments of MEH-PPV are also presented. The

  1. Characterization of Talbot pattern illumination for scanning optical microscopy

    NASA Astrophysics Data System (ADS)

    Liu, Guangshuo; Yang, Changhuei; Wu, Jigang

    2013-09-01

    We studied the use of Talbot pattern illumination in scanning optical microscopy (SOM). Unlike conventional illumination spots used in SOM, the focal spots in Talbot pattern are more complicated and do not have a simple Gaussian intensity distribution. To find out the resolution of SOM using Talbot pattern, we characterized the evolution of the full-width-at-half-maximum spot size of the Talbot focal spots by computer simulation. We then simulated the SOM imaging under Talbot pattern illumination using the razor blade and the U.S. Air Force target as the sample objects, and compared the results with those performed with Gaussian spots as illumination. Using several foci searching algorithms, the optimal focal distances were found to be shorter than the theoretical Talbot distances. The simulation results were consistent with the experiment results published previously. We then provide a practical guidance for searching for optimal focal distances in the SOM based on these studies.

  2. Wavefront sensorless adaptive optics temporal focusing-based multiphoton microscopy

    PubMed Central

    Chang, Chia-Yuan; Cheng, Li-Chung; Su, Hung-Wei; Hu, Yvonne Yuling; Cho, Keng-Chi; Yen, Wei-Chung; Xu, Chris; Dong, Chen Yuan; Chen, Shean-Jen

    2014-01-01

    Temporal profile distortions reduce excitation efficiency and image quality in temporal focusing-based multiphoton microscopy. In order to compensate the distortions, a wavefront sensorless adaptive optics system (AOS) was integrated into the microscope. The feedback control signal of the AOS was acquired from local image intensity maximization via a hill-climbing algorithm. The control signal was then utilized to drive a deformable mirror in such a way as to eliminate the distortions. With the AOS correction, not only is the axial excitation symmetrically refocused, but the axial resolution with full two-photon excited fluorescence (TPEF) intensity is also maintained. Hence, the contrast of the TPEF image of a R6G-doped PMMA thin film is enhanced along with a 3.7-fold increase in intensity. Furthermore, the TPEF image quality of 1μm fluorescent beads sealed in agarose gel at different depths is improved. PMID:24940539

  3. Optical-resolution photoacoustic microscopy of ischemic stroke

    NASA Astrophysics Data System (ADS)

    Hu, Song; Gonzales, Ernie; Soetikno, Brian; Gong, Enhao; Yan, Ping; Maslov, Konstantin; Lee, Jin-Moo; Wang, Lihong V.

    2011-03-01

    A major obstacle in understanding the mechanism of ischemic stroke is the lack of a tool to noninvasively or minimally invasively monitor cerebral hemodynamics longitudinally. Here, we applied optical-resolution photoacoustic microscopy (OR-PAM) to longitudinally study ischemic stroke induced brain injury in a mouse model with transient middle cerebral artery occlusion (MCAO). OR-PAM showed that, during MCAO, the average hemoglobin oxygen saturation (sO2) values of feeder arteries and draining veins within the stroke core region dropped ~10% and ~34%, respectively. After reperfusion, arterial sO2 recovered back to the baseline; however, the venous sO2 increased above the baseline value by ~7%. Thereafter, venous sO2 values were close to the arterial sO2 values, suggesting eventual brain tissue infarction.

  4. Optical pump-probe microscopy for biomedicine and art conservation

    NASA Astrophysics Data System (ADS)

    Fischer, Martin

    2013-03-01

    Nonlinear optical microscopy can provide contrast in highly heterogeneous media and a wide range of applications has emerged, primarily in biology, medicine, and materials science. Compared to linear microscopy methods, the localized nature of nonlinear interactions leads to high spatial resolution, optical sectioning, and larger possible imaging depth in scattering media. However, nonlinear contrast (other than fluorescence, harmonic generation or CARS) is generally difficult to measure because it is overwhelmed by the large background of detected illumination light. This background can be suppressed by using femtosecond pulse or pulse train shaping to encode nonlinear interactions in background-free regions of the frequency spectrum. We have developed this shaping technology to study novel intrinsic structural and molecular contrast in biological tissue, generally using less power than a laser pointer. For example we have recently been able to sensitively measure detailed transient absorption dynamics of melanin sub-types in a variety of skin lesions, showing clinically relevant differences of melanin type and distribution between cancerous and benign tissue.[1] Recently we have also applied this technology to paint samples and to historic artwork in order to provide detailed, depth-resolved pigment identification. Initial studies in different inorganic and organic pigments have shown a rich and pigment-specific nonlinear absorption signature.[2] Some pigments, for example lapis lazuli (natural ultramarine), even show marked differences in signal depending on its geographic origin and on age, demonstrating the potential of this technique to determine authenticity, provenance, technology of manufacture, or state of preservation of historic works of art.

  5. Combined conjugate and pupil adaptive optics in widefield microscopy

    NASA Astrophysics Data System (ADS)

    Beaulieu, Devin R.

    Traditionally, adaptive optics (AO) systems for microscopy have focused on AO at the pupil plane, however this produces poor performance in samples with both spatially-variant aberrations, such as non-flat sample interfaces, and spatially-invariant aberrations, such as spherical aberration due to a difference between the sample index of refraction and the sample for which the objective was designed. Here, we demonstrate well-corrected, wide field-of-view (FOV) microscopy by simultaneously correcting the two types of aberrations using two AO loops. Such an approach is necessary in wide-field applications where both types of aberration may be present, as each AO loop can only fully correct one type of aberration. Wide FOV corrections are demonstrated in a trans-illumination microscope equipped with two deformable mirrors (DMs), using a partitioned aperture wavefront (PAW) sensor to directly control the DM conjugated to the sample interface and a sensor-less genetic algorithm to control the DM conjugated to the objective's pupil.

  6. Extended focus Fourier domain optical coherence microscopy assists developmental biology

    NASA Astrophysics Data System (ADS)

    Villiger, Martin L.; Beleut, Manfred; Brisken, Cathrin; Lasser, Theo; Leitgeb, Rainer A.

    2007-07-01

    We present a novel detection scheme for Fourier domain optical coherence microscopy (FDOCM). A Bessel-like interference pattern with a strong central lobe was created with an axicon lens. This pattern was then imaged by a telescopic system into the sample space to obtain a laterally highly confined illumination needle, extending over a long axial range. For increased efficiency, the detection occurs decoupled from the illumination, avoiding a double pass through the axicon. Nearly constant transverse resolution of ~1.5μm along a focal range of 200μm with a maximum sensitivity of 105dB was obtained. A broad bandwidth Ti:Sapphire laser allowed for an axial resolution of 3μm in air, providing the nearly isotropic resolution necessary to access the microstructure of biological tissues. Together with the speed- and sensitivity-advantage of FDOCT, this system can perform in vivo measurements in a minimally invasive way. Tomograms of the mouse mammary gland and the mouse follicle, recorded in vitro, revealed biologically relevant structural details. Images acquired with classical microscopy techniques, involving stained and fluorescent samples, validate these structures and emphasize the high contrast of the tomograms. It is comparable to the contrast achieved with classical techniques, but employing neither staining, labeling nor slicing of the samples, stressing the high potential of FDOCM for minimally invasive in vivo small animal imaging.

  7. Multimodal nonlinear optical microscopy used to discriminate human colon cancer

    NASA Astrophysics Data System (ADS)

    Adur, Javier; Pelegati, Vitor B.; Bianchi, Mariana; de Thomaz, André A.; Baratti, Mariana O.; Carvalho, Hernandes F.; Casco, Víctor H.; Cesar, Carlos L.

    2013-02-01

    Colon cancer is one of the most diffused cancers in the Western World, ranking third worldwide in frequency of incidence after lung and breast cancers. Even if it is curable when detected and treated early, a more accurate premature diagnosis would be a suitable aim for both cancer prognostic and treatment. Combined multimodal nonlinear optical (NLO) microscopies, such as two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third harmonic generation (THG), and fluorescence lifetime imaging microscopy (FLIM) can be used to detect morphological and metabolic changes associated with stroma and epithelial transformation in colon cancer disease. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns between normal and malignant human colonic mucosa. Using a set of scoring methods significant differences both in the content, distribution and organization of stroma collagen fibrils, and lifetime components of NADH and FAD cofactors of human colon mucosa biopsies were found. Our results provide a framework for using NLO techniques as a clinical diagnostic tool for human colon cancer, and also suggest that the SHG and FLIM metrics could be applied to other intestinal disorders, which are characterized by abnormal cell proliferation and collagen assembly.

  8. New fluorinated rhodamines for optical microscopy and nanoscopy.

    PubMed

    Mitronova, Gyuzel Yu; Belov, Vladimir N; Bossi, Mariano L; Wurm, Christian A; Meyer, Lars; Medda, Rebecca; Moneron, Gael; Bretschneider, Stefan; Eggeling, Christian; Jakobs, Stefan; Hell, Stefan W

    2010-04-19

    New photostable rhodamine dyes represented by the compounds 1 a-r and 3-5 are proposed as efficient fluorescent markers with unique combination of structural features. Unlike rhodamines with monoalkylated nitrogen atoms, N',N-bis(2,2,2-trifluoroethyl) derivatives 1 e, 1 i, 1 j, 3-H and 5 were found to undergo sulfonation of the xanthene fragment at the positions 4' and 5'. Two fluorine atoms were introduced into the positions 2' and 7' of the 3',6'-diaminoxanthene fragment in compounds 1 a-d, 1 i-l and 1 m-r. The new rhodamine dyes may be excited with λ=488 or 514 nm light; most of them emit light at λ=512-554 nm (compounds 1 q and 1r at λ=576 and 589 nm in methanol, respectively) and have high fluorescence quantum yields in solution (up to 98 %), relatively long excited-state lifetimes (>3 ns) and are resistant against photobleaching, especially at high laser intensities, as is usually applied in confocal microscopy. Sulfonation of the xanthene fragment with 30 % SO3 in H2SO4 is compatible with the secondary amide bond (rhodamine-CON(Me)CH2CH2COOH) formed with MeNHCH2CH2COOCH3 to providing the sterically unhindered carboxylic group required for further (bio)conjugation reactions. After creating the amino reactive sites, the modified derivatives may be used as fluorescent markers and labels for (bio)molecules in optical microscopy and nanoscopy with very-high light intensities. Further, the new rhodamine dyes are able to pass the plasma membrane of living cells, introducing them as potential labels for recent live-cell-tag approaches. We exemplify the excellent performance of the fluorinated rhodamines in optical microscopy by fluorescence correlation spectroscopy (FCS) and stimulated emission depletion (STED) nanoscopy experiments. PMID:20309973

  9. Sample heating in near-field scanning optical microscopy

    NASA Astrophysics Data System (ADS)

    Erickson, Elizabeth S.; Dunn, Robert C.

    2005-11-01

    Heating near the aperture of aluminum coated, fiber optic near-field scanning optical microscopy probes was studied as a function of input and output powers. Using the shear-force feedback method, near-field probes were positioned nanometers above a thermochromic polymer and spectra were recorded as the input power was varied. Excitation at 405 nm of a thin polymer film incorporating perylene and N-allyl-N-methylaniline leads to dual emission peaks in the spectra. The relative peak intensity is temperature sensitive leading to a ratiometric measurement, which avoids complications based solely on intensity. Using this method, we find that the proximal end of typical near-field probes modestly increase in temperature to 40-45 °C at output powers of a few nanowatts (input power of ˜0.15mW). This increases to 55-65 °C at higher output powers of 50 nW or greater (input power of ˜2-4mW). Thermal heating of the probe at higher powers leads to probe elongation, which limits the heating experienced by the sample.

  10. Full-color structured illumination optical sectioning microscopy

    PubMed Central

    Qian, Jia; Lei, Ming; Dan, Dan; Yao, Baoli; Zhou, Xing; Yang, Yanlong; Yan, Shaohui; Min, Junwei; Yu, Xianghua

    2015-01-01

    In merits of super-resolved resolution and fast speed of three-dimensional (3D) optical sectioning capability, structured illumination microscopy (SIM) has found variety of applications in biomedical imaging. So far, most SIM systems use monochrome CCD or CMOS cameras to acquire images and discard the natural color information of the specimens. Although multicolor integration scheme are employed, multiple excitation sources and detectors are required and the spectral information is limited to a few of wavelengths. Here, we report a new method for full-color SIM with a color digital camera. A data processing algorithm based on HSV (Hue, Saturation, and Value) color space is proposed, in which the recorded color raw images are processed in the Hue, Saturation, Value color channels, and then reconstructed to a 3D image with full color. We demonstrated some 3D optical sectioning results on samples such as mixed pollen grains, insects, micro-chips and the surface of coins. The presented technique is applicable to some circumstance where color information plays crucial roles, such as in materials science and surface morphology. PMID:26415516

  11. Full-color structured illumination optical sectioning microscopy

    NASA Astrophysics Data System (ADS)

    Qian, Jia; Lei, Ming; Dan, Dan; Yao, Baoli; Zhou, Xing; Yang, Yanlong; Yan, Shaohui; Min, Junwei; Yu, Xianghua

    2015-09-01

    In merits of super-resolved resolution and fast speed of three-dimensional (3D) optical sectioning capability, structured illumination microscopy (SIM) has found variety of applications in biomedical imaging. So far, most SIM systems use monochrome CCD or CMOS cameras to acquire images and discard the natural color information of the specimens. Although multicolor integration scheme are employed, multiple excitation sources and detectors are required and the spectral information is limited to a few of wavelengths. Here, we report a new method for full-color SIM with a color digital camera. A data processing algorithm based on HSV (Hue, Saturation, and Value) color space is proposed, in which the recorded color raw images are processed in the Hue, Saturation, Value color channels, and then reconstructed to a 3D image with full color. We demonstrated some 3D optical sectioning results on samples such as mixed pollen grains, insects, micro-chips and the surface of coins. The presented technique is applicable to some circumstance where color information plays crucial roles, such as in materials science and surface morphology.

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

    NASA Astrophysics Data System (ADS)

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

    2009-07-01

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

  13. Non-iterative adaptive optical microscopy using wavefront sensing

    NASA Astrophysics Data System (ADS)

    Tao, X.; Azucena, O.; Kubby, J.

    2016-03-01

    This paper will review the development of wide-field and confocal microscopes with wavefront sensing and adaptive optics for correcting refractive aberrations and compensating scattering when imaging through thick tissues (Drosophila embryos and mouse brain tissue). To make wavefront measurements in biological specimens we have modified the laser guide-star techniques used in astronomy for measuring wavefront aberrations that occur as star light passes through Earth's turbulent atmosphere. Here sodium atoms in Earth's mesosphere, at an altitude of 95 km, are excited to fluoresce at resonance by a high-power sodium laser. The fluorescent light creates a guide-star reference beacon at the top of the atmosphere that can be used for measuring wavefront aberrations that occur as the light passes through the atmosphere. We have developed a related approach for making wavefront measurements in biological specimens using cellular structures labeled with fluorescent proteins as laser guide-stars. An example is a fluorescently labeled centrosome in a fruit fly embryo or neurons and dendrites in mouse brains. Using adaptive optical microscopy we show that the Strehl ratio, the ratio of the peak intensity of an aberrated point source relative to the diffraction limited image, can be improved by an order of magnitude when imaging deeply into live dynamic specimens, enabling near diffraction limited deep tissue imaging.

  14. DVD pickup head based optical resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Po-Hsun; Li, Meng-Lin

    2012-02-01

    Optical resolution photoacoustic microscopy (OR-PAM) has been shown as a promising tool for label-free microvascular and single-cell imaging in clinical and bioscientific applications. However, most OR-PAM systems are realized by using a bulky laser for photoacoustic excitation. The large volume and high price of the laser may restrain the popularity of OR-PAM. In this study, we develop a low-cost and compact OR-PAM system based on a commercially available DVD pickup head. We showed that the DVD pickup head have the required laser energy and focusing optics for OR-PAM. The firmware of a DVD burner was modified to enable its laser diode to provide a 13-ns laser pulse with 1.3-nJ energy at 650 nm. Two excitation wavelengths at 650 and 780 nm were available. The laser beam was focused onto the target after passing through a 0.6-mm thick DVD transparent polycarbonate coating, and then aligned to be confocal with a 50-MHz focused ultrasonic transducer in forward mode. To keep the target on focus, a scan involving auto-tracking procedure was performed. The lateral resolution was verified via cross-sectional imaging of a 6-μm carbon fiber. The measured -6 dB width of the carbon fiber was 6.66 μm which was in agreement with optical diffraction limit. The proposed OR-PAM has potential as an economically viable and compact blood screening tool available outside of large laboratories due to its low cost and portability. Furthermore, a better spatial resolution could be provided by using a blue ray DVD pickup head.

  15. Optical coherence microscopy of mouse cortical vasculature surrounding implanted electrodes

    NASA Astrophysics Data System (ADS)

    Hammer, Daniel X.; Lozzi, Andrea; Abliz, Erkinay; Greenbaum, Noah; Turner, Kevin P.; Pfefer, T. Joshua; Agrawal, Anant; Krauthamer, Victor; Welle, Cristin G.

    2014-03-01

    Optical coherence microscopy (OCM) provides real-time, in-vivo, three-dimensional, isotropic micron-resolution structural and functional characterization of tissue, cells, and other biological targets. Optical coherence angiography (OCA) also provides visualization and quantification of vascular flow via speckle-based or phase-resolved techniques. Performance assessment of neuroprosthetic systems, which allow direct thought control of limb prostheses, may be aided by OCA. In particular, there is a need to examine the underlying mechanisms of chronic functional degradation of implanted electrodes. Angiogenesis, capillary network remodeling, and changes in flow velocity are potential indicators of tissue changes that may be associated with waning electrode performance. The overall goal of this investigation is to quantify longitudinal changes in vascular morphology and capillary flow around neural electrodes chronically implanted in mice. We built a 1315-nm OCM system to image vessels in neocortical tissue in a cohort of mice. An optical window was implanted on the skull over the primary motor cortex above a penetrating shank-style microelectrode array. The mice were imaged bi-weekly to generate vascular maps of the region surrounding the implanted microelectrode array. Acute effects of window and electrode implantation included vessel dilation and profusion of vessels in the superficial layer of the cortex (0-200 μm). In deeper layers surrounding the electrode, no qualitative differences were seen in this early phase. These measurements establish a baseline vascular tissue response from the cortical window preparation and lay the ground work for future longitudinal studies to test the hypothesis that vascular changes will be associated with chronic electrode degradation.

  16. Combined optical and mechanical scanning in optical-resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Li, Lei; Yeh, Chenghung; Hu, Song; Wang, Lidai; Soetikno, Brian T.; Chen, Ruimin; Zhou, Qifa; Shung, K. Kirk; Maslov, Konstantin I.; Wang, Lihong V.

    2014-03-01

    Combined optical and mechanical scanning (COMS) in optical-resolution photoacoustic microscopy (OR-PAM) has provided five scanning modes with fast imaging speed and wide field of view (FOV). With two-dimensional (2D) galvanometer-based optical scanning, we have achieved a 2 KHz B-scan rate and 50 Hz volumetric-scan rate, which enables real-time tracking of cell activities in vivo. With optical-mechanical hybrid 2D scanning, we are able to image a wide FOV (10×8 mm2) within 150 seconds, which is 20 times faster than the conventional mechanical scan in our second-generation OR-PAM. With three-dimensional mechanical-based contour scanning, we can maintain the optimal signal-to-noise ratio and spatial resolution of OR-PAM while imaging objects with uneven surfaces, which is ideal for fast and quantitative studies of tumors and the brain.

  17. Differential Polarization Nonlinear Optical Microscopy with Adaptive Optics Controlled Multiplexed Beams

    PubMed Central

    Samim, Masood; Sandkuijl, Daaf; Tretyakov, Ian; Cisek, Richard; Barzda, Virginijus

    2013-01-01

    Differential polarization nonlinear optical microscopy has the potential to become an indispensable tool for structural investigations of ordered biological assemblies and microcrystalline aggregates. Their microscopic organization can be probed through fast and sensitive measurements of nonlinear optical signal anisotropy, which can be achieved with microscopic spatial resolution by using time-multiplexed pulsed laser beams with perpendicular polarization orientations and photon-counting detection electronics for signal demultiplexing. In addition, deformable membrane mirrors can be used to correct for optical aberrations in the microscope and simultaneously optimize beam overlap using a genetic algorithm. The beam overlap can be achieved with better accuracy than diffraction limited point-spread function, which allows to perform polarization-resolved measurements on the pixel-by-pixel basis. We describe a newly developed differential polarization microscope and present applications of the differential microscopy technique for structural studies of collagen and cellulose. Both, second harmonic generation, and fluorescence-detected nonlinear absorption anisotropy are used in these investigations. It is shown that the orientation and structural properties of the fibers in biological tissue can be deduced and that the orientation of fluorescent molecules (Congo Red), which label the fibers, can be determined. Differential polarization microscopy sidesteps common issues such as photobleaching and sample movement. Due to tens of megahertz alternating polarization of excitation pulses fast data acquisition can be conveniently applied to measure changes in the nonlinear signal anisotropy in dynamically changing in vivo structures. PMID:24022688

  18. Refractive Optics for Hard X-ray Transmission Microscopy

    SciTech Connect

    Simon, M.; Last, A.; Mohr, J.; Nazmov, V.; Reznikova, E.; Ahrens, G.; Voigt, A.

    2011-09-09

    For hard x-ray transmission microscopy at photon energies higher than 15 keV we design refractive condenser and imaging elements to be used with synchrotron light sources as well as with x-ray tube sources. The condenser lenses are optimized for low x-ray attenuation--resulting in apertures greater than 1 mm--and homogeneous intensity distribution on the detector plane, whereas the imaging enables high-resolution (<100 nm) full-field imaging. To obtain high image quality at reasonable exposure times, custom-tailored matched pairs of condenser and imaging lenses are being developed. The imaging lenses (compound refractive lenses, CRLs) are made of SU-8 negative resist by deep x-ray lithography. SU-8 shows high radiation stability. The fabrication technique enables high-quality lens structures regarding surface roughness and arrangement precision with arbitrary 2D geometry. To provide point foci, crossed pairs of lenses are used. Condenser lenses have been made utilizing deep x-ray lithographic patterning of thick SU-8 layers, too, whereas in this case, the aperture is limited due to process restrictions. Thus, in terms of large apertures, condenser lenses made of structured and rolled polyimide film are more attractive. Both condenser types, x-ray mosaic lenses and rolled x-ray prism lenses (RXPLs), are considered to be implemented into a microscope setup. The x-ray optical elements mentioned above are characterized with synchrotron radiation and x-ray laboratory sources, respectively.

  19. Improved Detection Sensitivity of Line-Scanning Optical Coherence Microscopy

    PubMed Central

    Chen, Yu; Huang, Shu-Wei; Zhou, Chao; Potsaid, Benjamin; Fujimoto, James G.

    2012-01-01

    Optical coherence microscopy (OCM) is a promising technology for high-resolution cellular-level imaging in human tissues. Line-scanning OCM is a new form of OCM that utilizes line-field illumination for parallel detection. In this study, we demonstrate improved detection sensitivity by using an achromatic design for line-field generation. This system operates at 830-nm wavelength with 82-nm bandwidth. The measured axial resolution is 3.9 μm in air (corresponding to ~2.9 μm in tissue), and the transverse resolutions are 2.1 μm along the line-field illumination direction and 1.7 μm perpendicular to line illumination direction. The measured sensitivity is 98 dB with 25 line averages, resulting in an imaging speed of ~2 frames/s (516 lines/s). Real-time, cellular-level imaging of scattering tissues is demonstrated using human-colon specimens. PMID:22685379

  20. 3-D Optical Interference Microscopy at the Lateral Resolution

    NASA Astrophysics Data System (ADS)

    Lehmann, Peter; Niehues, Jan; Tereschenko, Stanislav

    2014-10-01

    For applications in micro- and nanotechnologies the lateral resolution of optical 3-D microscopes becomes an issue of increasing relevance. However, lateral resolution of 3-D microscopes is hard to define in a satisfying way. Therefore, we first study the measurement capabilities of a highly resolving white-light interference (WLI) microscope close to the limit of lateral resolution. Results of measurements and simulations demonstrate that better lateral resolution seems to be achievable based on the envelope evaluation of a WLI signal. Unfortunately, close to the lateral resolution limit errors in the measured amplitude of micro-structures appear. On the other hand, results of interferometric phase evaluation seem to be strongly low-pass filtered in this case. Furthermore, the instrument transfer characteristics and the lateral resolution capabilities of WLI instruments are also affected by polarization. TM polarized light is less sensitive to edge diffraction and thus systematic errors can be avoided. However, apart from ghost steps due to fringe order errors, the results of phase evaluation seem to be closer to the real surface topography if TE polarized light is used. The lateral resolution can be further improved by combining WLI and structured illumination microscopy. Since the measured height of rectangular profiles close to the lateral resolution limit is generally too small compared to the real height, we introduce a method based on phase evaluation which characterizes the heights of barely laterally resolved rectangular gratings correctly.

  1. All-optical photoacoustic microscopy using a MEMS scanning mirror

    NASA Astrophysics Data System (ADS)

    Chen, Sung-Liang; Xie, Zhixing; Ling, Tao; Wei, Xunbin; Guo, L. Jay; Wang, Xueding

    2013-03-01

    It has been studied that a potential marker to obtain prognostic information about bladder cancer is tumor neoangiogenesis, which can be quantified by morphometric characteristics such as microvascular density. Photoacoustic microscopy (PAM) can render sensitive three-dimensional (3D) mapping of microvasculature, providing promise to evaluate the neoangiogenesis that is closely related to the diagnosis of bladder cancer. To ensure good image quality, it is desired to acquire bladder PAM images from its inside via the urethra, like conventional cystoscope. Previously, we demonstrated all-optical PAM systems using polymer microring resonators to detect photoacoustic signals and galvanometer mirrors for laser scanning. In this work, we build a miniature PAM system using a microelectromechanical systems (MEMS) scanning mirror, demonstrating a prototype of an endoscopic PAM head capable of high imaging quality of the bladder. The system has high resolutions of 17.5 μm in lateral direction and 19 μm in the axial direction at a distance of 5.4 mm. Images of printed grids and the 3D structure of microvasculature in animal bladders ex vivo by the system are demonstrated.

  2. Absolute position total internal reflection microscopy with an optical tweezer

    PubMed Central

    Liu, Lulu; Woolf, Alexander; Rodriguez, Alejandro W.; Capasso, Federico

    2014-01-01

    A noninvasive, in situ calibration method for total internal reflection microscopy (TIRM) based on optical tweezing is presented, which greatly expands the capabilities of this technique. We show that by making only simple modifications to the basic TIRM sensing setup and procedure, a probe particle’s absolute position relative to a dielectric interface may be known with better than 10 nm precision out to a distance greater than 1 μm from the surface. This represents an approximate 10× improvement in error and 3× improvement in measurement range over conventional TIRM methods. The technique’s advantage is in the direct measurement of the probe particle’s scattering intensity vs. height profile in situ, rather than relying on assumptions, inexact system analogs, or detailed knowledge of system parameters for calibration. To demonstrate the improved versatility of the TIRM method in terms of tunability, precision, and range, we show our results for the hindered near-wall diffusion coefficient for a spherical dielectric particle. PMID:25512542

  3. Ex vivo imaging of human thyroid pathology using integrated optical coherence tomography and optical coherence microscopy

    NASA Astrophysics Data System (ADS)

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

    2010-01-01

    We evaluate the feasibility of optical coherence tomography (OCT) and optical coherence microscopy (OCM) for imaging of benign and malignant thyroid lesions ex vivo using intrinsic optical contrast. 34 thyroid gland specimens are imaged from 17 patients, covering a spectrum of pathology ranging from normal thyroid to benign disease/neoplasms (multinodular colloid goiter, Hashimoto's thyroiditis, and follicular adenoma) and malignant thyroid tumors (papillary carcinoma and medullary carcinoma). Imaging is performed using an integrated OCT and OCM system, with <4 μm axial resolution (OCT and OCM), and 14 μm (OCT) and <2 μm (OCM) transverse resolution. The system allows seamless switching between low and high magnifications in a way similar to traditional microscopy. Good correspondence is observed between optical images and histological sections. Characteristic features that suggest malignant lesions, such as complex papillary architecture, microfollicules, psammomatous calcifications, or replacement of normal follicular architecture with sheets/nests of tumor cells, can be identified from OCT and OCM images and are clearly differentiable from normal or benign thyroid tissues. With further development of needle-based imaging probes, OCT and OCM could be promising techniques to use for the screening of thyroid nodules and to improve the diagnostic specificity of fine needle aspiration evaluation.

  4. Through-focus scanning optical microscopy (TSOM) considering optical aberrations: practical implementation.

    PubMed

    Ryabko, Maxim; Shchekin, Alexey; Koptyaev, Sergey; Lantsov, Alexey; Medvedev, Anton; Shcherbakov, Alexander; Oh, Sang Yoon

    2015-12-14

    Through-focus scanning optical microscopy (TSOM) method based on use of a library, which is composed of simulated defocused images of nanosized silicon lines on the top of a monocrystalline silicon substrate, is demonstrated. The images are simulated using Finite-Differences in Time-Domain (FDTD) method taking into account optical aberrations of the experimental setup, which are measured experimentally. Consideration of the optical aberrations allows us to reduce the discrepancy between experimental and simulated defocused images of the samples under study to the value of ≈2%in contrast to ≈10% when the aberrations are not taken into account. It results in ≈5% recognition accuracy for critical dimension (CD) values in the range 40-150 nm. PMID:26699011

  5. Stent-induced coronary artery stenosis characterized by multimodal nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

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

    2011-02-01

    We demonstrate for the first time the applicability of multimodal nonlinear optical (NLO) microscopy to the interrogation of stented coronary arteries under different diet and stent deployment conditions. Bare metal stents and Taxus drug-eluting stents (DES) were placed in coronary arteries of Ossabaw pigs of control and atherogenic diet groups. Multimodal NLO imaging was performed to inspect changes in arterial structures and compositions after stenting. Sum frequency generation, one of the multimodalities, was used for the quantitative analysis of collagen content in the peristent and in-stent artery segments of both pig groups. Atherogenic diet increased lipid and collagen in peristent segments. In-stent segments showed decreased collagen expression in neointima compared to media. Deployment of DES in atheromatous arteries inhibited collagen expression in the arterial media.

  6. Near field scanning optical microscopy of polycrystalline semiconductors

    NASA Astrophysics Data System (ADS)

    Herndon, Mary Kay

    1999-09-01

    Photovoltaic devices are commonly used for space applications and remote terrestrial power requirements. Polycrystalline solar cell devices often have much lower efficiencies than their crystalline counterparts, but because they can be fabricated much more cheaply, they can still be cost-effective when compared to single crystal devices. The long term goal of this work is to provide information that will lead to higher quality devices with improved cost efficiency. In order to do this, a better understanding of the mechanisms that take place in these materials is needed. The goal of this thesis was to improve our understanding of these devices by adapting a novel characterization technique, Near Field Scanning Optical Microscopy (NSOM), to the study of polycrystalline films. Visible light NSOM is a relatively new technique that allows for optical characterization of materials with resolution beyond the far-field diffraction limit. By using NSOM to study the physical and electrical properties of polycrystalline solar cells, individual grains can be studied and more insight can be gained as to how various properties of the thin films affect the device efficiency. For this research, an NSOM was designed and built to be versatile enough to handle the sorts of samples and measurements required for studying a variety of photovoltaic devices. As a first step, the NSOM was used to characterize single crystal GaAs solar cell devices. Measurements of topography and NSOM-induced photocurrent were obtained simultaneously on cross sections of the material, allowing the p-n junction to be probed. Because the NSOM data could be compared to an expected result, this allowed verification of the new microscope's imaging capabilities and ensured accurate data interpretation. Effects of surface recombination were detected on the cleaved edges. The NSOM was used to characterize surface quality and study the effects of surface passivation treatments. Of the polycrystalline materials

  7. Automated interferometric synthetic aperture microscopy and computational adaptive optics for improved optical coherence tomography.

    PubMed

    Xu, Yang; Liu, Yuan-Zhi; Boppart, Stephen A; Carney, P Scott

    2016-03-10

    In this paper, we introduce an algorithm framework for the automation of interferometric synthetic aperture microscopy (ISAM). Under this framework, common processing steps such as dispersion correction, Fourier domain resampling, and computational adaptive optics aberration correction are carried out as metrics-assisted parameter search problems. We further present the results of this algorithm applied to phantom and biological tissue samples and compare with manually adjusted results. With the automated algorithm, near-optimal ISAM reconstruction can be achieved without manual adjustment. At the same time, the technical barrier for the nonexpert using ISAM imaging is also significantly lowered. PMID:26974799

  8. Simultaneous characterization of rotational and translational diffusion of optically anisotropic particles by optical microscopy

    NASA Astrophysics Data System (ADS)

    Giavazzi, Fabio; Haro-Pérez, Catalina; Cerbino, Roberto

    2016-05-01

    We probe the roto-translational Brownian motion of optically anisotropic particles suspended in water with a simple and straightforward optical microscopy experiment that does not require positional or rotational particle tracking. We acquire a movie of the suspension placed between two polarizing elements and we extract the translational diffusion coefficient D T and the rotational diffusion coefficient D R from the analysis of the temporal correlation properties of the spatial Fourier modes of the intensity fluctuations in the movie. Our method is successfully tested with a dilute suspension of birefringent spherical colloidal particles obtained by polymerizing an emulsion of droplets of liquid crystal in a nematic phase, whose roto-translational dynamics is found to be well described by theory. The simplicity of our approach makes our method a viable alternative to particle tracking and depolarized dynamic light scattering.

  9. Simultaneous characterization of rotational and translational diffusion of optically anisotropic particles by optical microscopy.

    PubMed

    Giavazzi, Fabio; Haro-Pérez, Catalina; Cerbino, Roberto

    2016-05-18

    We probe the roto-translational Brownian motion of optically anisotropic particles suspended in water with a simple and straightforward optical microscopy experiment that does not require positional or rotational particle tracking. We acquire a movie of the suspension placed between two polarizing elements and we extract the translational diffusion coefficient D T and the rotational diffusion coefficient D R from the analysis of the temporal correlation properties of the spatial Fourier modes of the intensity fluctuations in the movie. Our method is successfully tested with a dilute suspension of birefringent spherical colloidal particles obtained by polymerizing an emulsion of droplets of liquid crystal in a nematic phase, whose roto-translational dynamics is found to be well described by theory. The simplicity of our approach makes our method a viable alternative to particle tracking and depolarized dynamic light scattering. PMID:27093398

  10. Dynamic complex optical fields for optical manipulation, 3D microscopy, and photostimulation of neurotransmitters

    NASA Astrophysics Data System (ADS)

    Daria, Vincent R.; Stricker, Christian; Bekkers, John; Redman, Steve; Bachor, Hans

    2010-08-01

    We demonstrate a multi-functional system capable of multiple-site two-photon excitation of photo-sensitive compounds as well as transfer of optical mechanical properties on an array of mesoscopic particles. We use holographic projection of a single Ti:Sapphire laser operating in femtosecond pulse mode to show that the projected three-dimensional light patterns have sufficient spatiotemporal photon density for multi-site two-photon excitation of biological fluorescent markers and caged neurotransmitters. Using the same laser operating in continuous-wave mode, we can use the same light patterns for non-invasive transfer of both linear and orbital angular momentum on a variety of mesoscopic particles. The system also incorporates high-speed scanning using acousto-optic modulators to rapidly render 3D images of neuron samples via two-photon microscopy.

  11. Isolation of Optically Targeted Single Bacteria by Application of Fluidic Force Microscopy to Aerobic Anoxygenic Phototrophs from the Phyllosphere

    PubMed Central

    Stiefel, Philipp; Zambelli, Tomaso

    2013-01-01

    In their natural environment, bacteria often behave differently than they do under laboratory conditions. To gain insight into the physiology of bacteria in situ, dedicated approaches are required to monitor their adaptations and specific behaviors under environmental conditions. Optical microscopy is crucial for the observation of fundamental characteristics of bacteria, such as cell shape, size, and marker gene expression. Here, fluidic force microscopy (FluidFM) was exploited to isolate optically selected bacteria for subsequent identification and characterization. In this study, bacteriochlorophyll-producing bacteria, which can be visualized due to their characteristic fluorescence in the infrared range, were isolated from leaf washes. Bacterial communities from the phyllosphere were investigated because they harbor genes indicative of aerobic anoxygenic photosynthesis. Our data show that different species of Methylobacterium express their photosystem in planta, and they show a distinct pattern of bacteriochlorophyll production under laboratory conditions that is dependent on supplied carbon sources. PMID:23770907

  12. Multiscale imaging of human thyroid pathologies using integrated optical coherence tomography (OCT) and optical coherence microscopy (OCM)

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

    We evaluate the feasibility of optical coherence tomography (OCT) and optical coherence microscopy (OCM) for imaging of benign and malignant thyroid lesions ex vivo using intrinsic optical contrast. Thirty four thyroid gland specimens were imaged from 17 patients, covering a spectrum of pathology, ranging from normal thyroid to neoplasia and benign disease. The integrated OCT and OCM imaging system allows seamlessly switching between low and high magnifications, in a way similar to traditional microscopy. Good correspondence was observed between optical images and histological sections. The results provide a basis for interpretation of future OCT and OCM images of the thyroid tissues and suggest the possibility of future in vivo evaluation of thyroid pathology.

  13. An integrated optical coherence microscopy imaging and optical stimulation system for optogenetic pacing in Drosophila melanogaster (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Alex, Aneesh; Li, Airong; Men, Jing; Jerwick, Jason; Tanzi, Rudolph E.; Zhou, Chao

    2016-03-01

    Electrical stimulation is the clinical standard for cardiac pacing. Although highly effective in controlling cardiac rhythm, the invasive nature, non-specificity to cardiac tissues and possible tissue damage limits its applications. Optogenetic pacing of the heart is a promising alternative, which is non-invasive and more specific, has high spatial and temporal precision, and avoids the shortcomings in electrical stimulation. Drosophila melanogaster, which is a powerful model organism with orthologs of nearly 75% of human disease genes, has not been studied for optogenetic pacing in the heart. Here, we developed a non-invasive integrated optical pacing and optical coherence microscopy (OCM) imaging system to control the heart rhythm of Drosophila at different developmental stages using light. The OCM system is capable of providing high imaging speed (130 frames/s) and ultrahigh imaging resolutions (1.5 μm and 3.9 μm for axial and transverse resolutions, respectively). A light-sensitive pacemaker was developed in Drosophila by specifically expressing the light-gated cation channel, channelrhodopsin-2 (ChR2) in transgenic Drosophila heart. We achieved non-invasive and specific optical control of the Drosophila heart rhythm throughout the fly's life cycle (larva, pupa, and adult) by stimulating the heart with 475 nm pulsed laser light. Heart response to stimulation pulses was monitored non-invasively with OCM. This integrated non-invasive optogenetic control and in vivo imaging technique provides a novel platform for performing research studies in developmental cardiology.

  14. Automated Identification and Location Analysis of Marked Stem Cells Colonies in Optical Microscopy Images

    PubMed Central

    Paduano, Vincenzo; Tagliaferri, Daniela; Falco, Geppino; Ceccarelli, Michele

    2013-01-01

    Embryonic stem cells (ESCs) are characterized by two remarkable peculiarities: the capacity to propagate as undifferentiated cells (self-renewal) and the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives (pluripotency). Although the majority of ESCs divide without losing the pluripotency, it has become evident that ESC cultures consists of multiple cell populations highlighted by the expression of early germ lineage markers during spontaneous differentiation. Hence, the identification and characterization of ESCs subpopulations represents an efficient approach to improve the comprehension of correlation between gene expression and cell specification status. To study markers of ESCs heterogeneity, we developed an analysis pipeline which can automatically process images of stem cell colonies in optical microscopy. The question we try to address is to find out the statistically significant preferred locations of the marked cells. We tested our algorithm on a set of images of stem cell colonies to analyze the expression pattern of the Zscan4 gene, which was an elite candidate gene to be studied because it is specifically expressed in subpopulation of ESCs. To validate the proposed method we analyzed the behavior of control genes whose pattern had been associated to biological status such as differentiation (EndoA), pluripotency (Pou5f1), and pluripotency fluctuation (Nanog). We found that Zscan4 is not uniformly expressed inside a stem cell colony, and that it tends to be expressed towards the center of the colony, moreover cells expressing Zscan4 cluster each other. This is of significant importance because it allows us to hypothesize a biological status where the cells expressing Zscan4 are preferably associated to the inner of colonies suggesting pluripotent cell status features, and the clustering between themselves suggests either a colony paracrine effect or an early phase of cell specification through proliferation. Also, the analysis on

  15. Automated identification and location analysis of marked stem cells colonies in optical microscopy images.

    PubMed

    Paduano, Vincenzo; Tagliaferri, Daniela; Falco, Geppino; Ceccarelli, Michele

    2013-01-01

    Embryonic stem cells (ESCs) are characterized by two remarkable peculiarities: the capacity to propagate as undifferentiated cells (self-renewal) and the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives (pluripotency). Although the majority of ESCs divide without losing the pluripotency, it has become evident that ESC cultures consists of multiple cell populations highlighted by the expression of early germ lineage markers during spontaneous differentiation. Hence, the identification and characterization of ESCs subpopulations represents an efficient approach to improve the comprehension of correlation between gene expression and cell specification status. To study markers of ESCs heterogeneity, we developed an analysis pipeline which can automatically process images of stem cell colonies in optical microscopy. The question we try to address is to find out the statistically significant preferred locations of the marked cells. We tested our algorithm on a set of images of stem cell colonies to analyze the expression pattern of the Zscan4 gene, which was an elite candidate gene to be studied because it is specifically expressed in subpopulation of ESCs. To validate the proposed method we analyzed the behavior of control genes whose pattern had been associated to biological status such as differentiation (EndoA), pluripotency (Pou5f1), and pluripotency fluctuation (Nanog). We found that Zscan4 is not uniformly expressed inside a stem cell colony, and that it tends to be expressed towards the center of the colony, moreover cells expressing Zscan4 cluster each other. This is of significant importance because it allows us to hypothesize a biological status where the cells expressing Zscan4 are preferably associated to the inner of colonies suggesting pluripotent cell status features, and the clustering between themselves suggests either a colony paracrine effect or an early phase of cell specification through proliferation. Also, the analysis on

  16. Visualization of Mouse Neuronal Ganglia Infected by Herpes Simplex Virus 1 (HSV-1) Using Multimodal Non-Linear Optical Microscopy

    PubMed Central

    Rochette, Pierre-Alexandre; Laliberté, Mathieu; Bertrand-Grenier, Antony; Houle, Marie-Andrée; Blache, Marie-Claire; Légaré, François; Pearson, Angela

    2014-01-01

    Herpes simplex virus 1 (HSV-1) is a neurotropic virus that causes skin lesions and goes on to enter a latent state in neurons of the trigeminal ganglia. Following stress, the virus may reactivate from latency leading to recurrent lesions. The in situ study of neuronal infections by HSV-1 is critical to understanding the mechanisms involved in the biology of this virus and how it causes disease; however, this normally requires fixation and sectioning of the target tissues followed by treatment with contrast agents to visualize key structures, which can lead to artifacts. To further our ability to study HSV-1 neuropathogenesis, we have generated a recombinant virus expressing a second generation red fluorescent protein (mCherry), which behaves like the parental virus in vivo. By optimizing the application of a multimodal non-linear optical microscopy platform, we have successfully visualized in unsectioned trigeminal ganglia of mice both infected cells by two-photon fluorescence microscopy, and myelinated axons of uninfected surrounding cells by coherent anti-Stokes Raman scattering (CARS) microscopy. These results represent the first report of CARS microscopy being combined with 2-photon fluorescence microscopy to visualize virus-infected cells deep within unsectioned explanted tissue, and demonstrate the application of multimodal non-linear optical microscopy for high spatial resolution biological imaging of tissues without the use of stains or fixatives. PMID:25133579

  17. Advanced 3D Optical Microscopy in ENS Research.

    PubMed

    Vanden Berghe, Pieter

    2016-01-01

    Microscopic techniques are among the few approaches that have survived the test of time. Being invented half way the seventeenth century by Antonie van Leeuwenhoek and Robert Hooke, this technology is still essential in modern biomedical labs. Many microscopy techniques have been used in ENS research to guide researchers in their dissections and later to enable electrode recordings. Apart from this, microscopy has been instrumental in the identification of subpopulations of cells in the ENS, using a variety of staining methods. A significant step forward in the use of microscopy was the introduction of fluorescence approaches. Due to the fact that intense excitation light is now filtered away from the longer wavelength emission light, the contrast can be improved drastically, which helped to identify subpopulations of enteric neurons in a variety of species. Later functionalized fluorescent probes were used to measure and film activity in muscle and neuronal cells. Another important impetus to the use of microscopy was the discovery and isolation of the green fluorescent protein (GFP), as it gave rise to the development of many different color variants and functionalized constructs. Recent advances in microscopy are the result of a continuous search to enhance contrast between the item of interest and its background but also to improve resolving power to tell two small objects apart. In this chapter three different microscopy approaches will be discussed that can aid to improve our understanding of ENS function within the gut wall. PMID:27379646

  18. Nonlinear optical microscopy in biology: Combining second-harmonic generation and two-photon fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Clays, Koen

    2011-03-01

    Optical microscopy has been since long a truly enabling visualization technique in the biological and biomedical sciences. Linear optical microscopy relies on simple linear optical effects. Nonlinear optical microscopy relies on the nonlinear optical properties of endogenous or exogenous chromophores to produce a better image. Two-photon fluorescence (TPF), a third-order nonlinear optical effect and observed at the focal spot only due to the quadratic intensity dependence, results in inherently higher resolution than possible for one-photon fluorescence, observed over the complete Rayleigh range. Second-harmonic generation (SHG) is a second-order nonlinear optical effect only observed for non-centrosymmetric arrangements of non-centrosymmetric chromophores. While this does put a restriction on the chromophores that can be used, it also results in structural information about symmetry when used in combination with TPF. TPF, being a third-order nonlinear process, is not restricted by any symmetry consideration. We will review the molecular design criteria for exogenous probes for combined SHG and TPF nonlinear microscopy, provide examples of optimized chromophores and show microscopy images demonstrating the use of such chromophores in nonlinear microscopy.

  19. Polarization Properties in Apertureless-Type Scanning Near-Field Optical Microscopy.

    PubMed

    Ishibashi, Takayuki; Cai, Yongfu

    2015-12-01

    Polarization properties of apertureless-type scanning near-field optical microscopy (a-SNOM) were measured experimentally and were also analyzed using a finite-difference time-domain (FDTD) simulation. Our study reveals that the polarization properties in the a-SNOM are maintained and the a-SNOM works as a wave plate expressed by a Jones matrix. The measured signals obtained by the lock-in detection technique could be decomposed into signals scattered from near-field region and background signals reflected by tip and sample. Polarization images measured by a-SNOM with an angle resolution of 1° are shown. FDTD analysis also reveals the polarization properties of light in the area between a tip and a sample are p-polarization in most of cases. PMID:26415540

  20. High precision deflection measurement of microcantilever in an optical pickup head based atomic force microscopy

    SciTech Connect

    Lee, Sang Heon

    2012-11-15

    This paper presents the methodology to measure the precise deflection of microcantilever in an optical pickup head based atomic force microscopy. In this paper, three types of calibration methods have been proposed: full linearization, sectioned linearization, and the method based on astigmatism. In addition, the probe heads for easy calibration of optical pickup head and fast replacement of optical pickup head have been developed. The performances of each method have been compared through a set of experiments and constant height mode operation which was not possible in the optical pickup head based atomic force microscopy has been carried out successfully.

  1. Gold Coating of Fiber Tips in Near-Field Scanning Optical Microscopy

    NASA Technical Reports Server (NTRS)

    Vikram, Chandra S.; Witherow, William K.

    2000-01-01

    We report what is believed to be the first experimental demonstration of gold coating by a chemical baking process on tapered fiber tips used in near-field scanning optical microscopy. Many tips can be simultaneously coated.

  2. Subwavelength-resolution photoacoustic microscopy for label-free detection of optical absorption in vivo

    NASA Astrophysics Data System (ADS)

    Zhang, Chi; Maslov, Konstantin; Wang, Lihong V.

    2011-03-01

    Mainstream optical microscopy technologies normally detect fluorescence or scattering, which may require undesirable labeling, but cannot directly sense optical absorption, which provides essential biological functional information. Here we reported in vivo and label-free subwavelength-resolution photoacoustic microscopy (SW-PAM) by using a waterimmersion optical objective with a 1.23 NA. Capable of detecting nonfluorescent endogenous pigments, SW-PAM provides exquisitely high optical-absorption contrast. And, as a result of background-free detection, the sensitivity of SW-PAM to optical absorption reaches 100%. SW-PAM was demonstrated with wide-field optical microscopy by imaging gold nanospheres, ex vivo cells, and in vivo vasculature and melanoma. It was shown that SW-PAM has approached the ultimate diffraction-limited optical resolution-220 nm resolution at 532 nm wavelength. Subcellular organelles, such as melanosomes, can be resolved by SW-PAM. Vasculature and early-stage melanoma were imaged with 21:1 and 34:1 contrasts, respectively, without labeling. For all these applications, SW-PAM has contrasts orders of magnitude higher than wide-field optical microscopy. Therefore, SW-PAM is expected to join the mainstream microscopy technologies.

  3. GPU-based computational adaptive optics for volumetric optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Tang, Han; Mulligan, Jeffrey A.; Untracht, Gavrielle R.; Zhang, Xihao; Adie, Steven G.

    2016-03-01

    Optical coherence tomography (OCT) is a non-invasive imaging technique that measures reflectance from within biological tissues. Current higher-NA optical coherence microscopy (OCM) technologies with near cellular resolution have limitations on volumetric imaging capabilities due to the trade-offs between resolution vs. depth-of-field and sensitivity to aberrations. Such trade-offs can be addressed using computational adaptive optics (CAO), which corrects aberration computationally for all depths based on the complex optical field measured by OCT. However, due to the large size of datasets plus the computational complexity of CAO and OCT algorithms, it is a challenge to achieve high-resolution 3D-OCM reconstructions at speeds suitable for clinical and research OCM imaging. In recent years, real-time OCT reconstruction incorporating both dispersion and defocus correction has been achieved through parallel computing on graphics processing units (GPUs). We add to these methods by implementing depth-dependent aberration correction for volumetric OCM using plane-by-plane phase deconvolution. Following both defocus and aberration correction, our reconstruction algorithm achieved depth-independent transverse resolution of 2.8 um, equal to the diffraction-limited focal plane resolution. We have translated the CAO algorithm to a CUDA code implementation and tested the speed of the software in real-time using two GPUs - NVIDIA Quadro K600 and Geforce TITAN Z. For a data volume containing 4096×256×256 voxels, our system's processing speed can keep up with the 60 kHz acquisition rate of the line-scan camera, and takes 1.09 seconds to simultaneously update the CAO correction for 3 en face planes at user-selectable depths.

  4. Noninvasive determination of optical lever sensitivity in atomic force microscopy

    SciTech Connect

    Higgins, M.J.; Proksch, R.; Sader, J.E.; Polcik, M.; Mc Endoo, S.; Cleveland, J.P.; Jarvis, S.P.

    2006-01-15

    Atomic force microscopes typically require knowledge of the cantilever spring constant and optical lever sensitivity in order to accurately determine the force from the cantilever deflection. In this study, we investigate a technique to calibrate the optical lever sensitivity of rectangular cantilevers that does not require contact to be made with a surface. This noncontact approach utilizes the method of Sader et al. [Rev. Sci. Instrum. 70, 3967 (1999)] to calibrate the spring constant of the cantilever in combination with the equipartition theorem [J. L. Hutter and J. Bechhoefer, Rev. Sci. Instrum. 64, 1868 (1993)] to determine the optical lever sensitivity. A comparison is presented between sensitivity values obtained from conventional static mode force curves and those derived using this noncontact approach for a range of different cantilevers in air and liquid. These measurements indicate that the method offers a quick, alternative approach for the calibration of the optical lever sensitivity.

  5. Integrated optical- and acoustic-resolution photoacoustic microscopy based on an optical fiber bundle

    PubMed Central

    Maslov, Konstantin; Wang, Lihong V.

    2014-01-01

    Photoacoustic microscopy (PAM), whose spatial resolution and maximum imaging depth are both scalable, has made great progress in recent years. However, each PAM system currently achieves only one resolution with an associated maximum imaging depth. Here, we present an integrated optical-resolution (OR) and acoustic-resolution (AR) PAM system implemented by delivering light via an optical fiber bundle. A single fiber core is used to deliver light for OR illumination in order to achieve a small spot size and hence high lateral resolution, whereas all the fiber cores are used to deliver more energy for AR illumination. Most other components are shared by the OR and AR imaging. The lateral resolution can be seamlessly switched between 2.2 μm and 40 μm as the maximum imaging depth is switched between 1.3 mm and 3.0 mm. The system enables automatically co-registered higher-resolution OR and deeper AR photoacoustic imaging. PMID:23282835

  6. Sensorless adaptive optics and the effect of field of view in biological second harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Vandendriessche, Stefaan; Vanbel, Maarten K.; Verbiest, Thierry

    2014-05-01

    In light of the population aging in many developed countries, there is a great economical interest in improving the speed and cost-efficiency of healthcare. Clinical diagnosis tools are key to these improvements, with biophotonics providing a means to achieve them. Standard optical microscopy of in vitro biological samples has been an important diagnosis tool since the invention of the microscope, with well known resolution limits. Nonlinear optical imaging improves on the resolution limits of linear microscopy, while providing higher contrast images and a greater penetration depth due to the red-shifted incident light compared to standard optical microscopy. It also provides information on molecular orientation and chirality. Adaptive optics can improve the quality of nonlinear optical images. We analyzed the effect of sensorless adaptive optics on the quality of the nonlinear optical images of biological samples. We demonstrate that care needs to be taken when using a large field of view. Our findings provide information on how to improve the quality of nonlinear optical imaging, and can be generalized to other in vitro biological samples. The image quality improvements achieved by adaptive optics should help speed up clinical diagnostics in vitro, while increasing their accuracy and helping decrease detection limits. The same principles apply to in vivo biological samples, and in the future it may be possible to extend these findings to other nonlinear optical effects used in biological imaging.

  7. All-optical optoacoustic microscopy system based on probe beam deflection technique

    NASA Astrophysics Data System (ADS)

    Maswadi, Saher M.; Tsyboulskic, Dmitri; Roth, Caleb C.; Glickman, Randolph D.; Beier, Hope T.; Oraevsky, Alexander A.; Ibey, Bennett L.

    2016-03-01

    It is difficult to achieve sub-micron resolution in backward mode OA microscopy using conventional piezoelectric detectors, because of wavefront distortions caused by components placed in the optical path, between the sample and the objective lens, that are required to separate the acoustic wave from the optical beam. As an alternate approach, an optoacoustic microscope (OAM) was constructed using the probe beam deflection technique (PBDT) to detect laserinduced acoustic signals. The all-optical OAM detects laser-generated pressure waves using a probe beam passing through a coupling medium, such as water, filling the space between the microscope objective lens and sample. The acoustic waves generated in the sample propagate through the coupling medium, causing transient changes in the refractive index that deflect the probe beam. These deflections are measured with a high-speed, balanced photodiode position detector. The deflection amplitude is directly proportional to the magnitude of the acoustic pressure wave, and provides the data required for image reconstruction. The sensitivity of the PBDT detector expressed as noise equivalent pressure was 12 Pa, comparable to that of existing high-performance ultrasound detectors. Because of the unimpeded working distance, a high numerical aperture objective lens, i.e. NA = 1, was employed in the OAM to achieve near diffraction-limited lateral resolution of 0.5 μm at 532nm. The all-optical OAM provides several benefits over current piezoelectric detector-based systems, such as increased lateral and axial resolution, higher sensitivity, robustness, and potentially more compatibility with multimodal instruments.

  8. Confocal Microscopy and Molecular-Specific Optical Contrast Agents for the Detection of Oral Neoplasia

    PubMed Central

    Carlson, Alicia L.; Gillenwater, Ann M.; Williams, Michelle D.; El-Naggar, Adel K.; Richards-Kortum, R. R.

    2009-01-01

    Using current clinical diagnostic techniques, it is difficult to visualize tumor morphology and architecture at the cellular level, which is necessary for diagnostic localization of pathologic lesions. Optical imaging techniques have the potential to address this clinical need by providing real-time, sub-cellular resolution images. This paper describes the use of dual mode confocal microscopy and optical molecular-specific contrast agents to image tissue architecture, cellular morphology, and sub-cellular molecular features of normal and neoplastic oral tissues. Fresh tissue slices were prepared from 33 biopsies of clinically normal and abnormal oral mucosa obtained from 14 patients. Reflectance confocal images were acquired after the application of 6% acetic acid, and fluorescence confocal images were acquired after the application of a fluorescence contrast agent targeting the epidermal growth factor receptor (EGFR). The dual imaging modes provided images similar to light microscopy of hematoxylin and eosin and immunohistochemistry staining, but from thick fresh tissue slices. Reflectance images provided information on the architecture of the tissue and the cellular morphology. The nuclear-to-cytoplasmic (N/C) ratio from the reflectance images was at least 7.5 times greater for the carcinoma than the corresponding normal samples, except for one case of highly keratinized carcinoma. Separation of carcinoma from normal and mild dysplasia was achieved using this ratio (p<0.01). Fluorescence images of EGFR expression yielded a mean fluorescence labeling intensity (FLI) that was at least 2.7 times higher for severe dysplasia and carcinoma samples than for the corresponding normal sample, and could be used to distinguish carcinoma from normal and mild dysplasia (p<0.01). Analyzed together, the N/C ratio and the mean FLI may improve the ability to distinguish carcinoma from normal squamous epithelium. PMID:17877424

  9. Accurate cell counts in live mouse embryos using optical quadrature and differential interference contrast microscopy

    NASA Astrophysics Data System (ADS)

    Warger, William C., II; Newmark, Judith A.; Zhao, Bing; Warner, Carol M.; DiMarzio, Charles A.

    2006-02-01

    Present imaging techniques used in in vitro fertilization (IVF) clinics are unable to produce accurate cell counts in developing embryos past the eight-cell stage. We have developed a method that has produced accurate cell counts in live mouse embryos ranging from 13-25 cells by combining Differential Interference Contrast (DIC) and Optical Quadrature Microscopy. Optical Quadrature Microscopy is an interferometric imaging modality that measures the amplitude and phase of the signal beam that travels through the embryo. The phase is transformed into an image of optical path length difference, which is used to determine the maximum optical path length deviation of a single cell. DIC microscopy gives distinct cell boundaries for cells within the focal plane when other cells do not lie in the path to the objective. Fitting an ellipse to the boundary of a single cell in the DIC image and combining it with the maximum optical path length deviation of a single cell creates an ellipsoidal model cell of optical path length deviation. Subtracting the model cell from the Optical Quadrature image will either show the optical path length deviation of the culture medium or reveal another cell underneath. Once all the boundaries are used in the DIC image, the subtracted Optical Quadrature image is analyzed to determine the cell boundaries of the remaining cells. The final cell count is produced when no more cells can be subtracted. We have produced exact cell counts on 5 samples, which have been validated by Epi-Fluorescence images of Hoechst stained nuclei.

  10. A Minimal Optical Trapping and Imaging Microscopy System

    PubMed Central

    Hernández Candia, Carmen Noemí; Tafoya Martínez, Sara; Gutiérrez-Medina, Braulio

    2013-01-01

    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. PMID:23451216

  11. Optical Microscopy Techniques to Inspect for Metallic Whiskers

    NASA Technical Reports Server (NTRS)

    Brusse, Jay A.

    2006-01-01

    Metal surface finishes of tin, zinc and cadmium are often applied to electronic components, mechanical hardware and other structures. These finishes sometimes unpredictably may form metal whiskers over periods that can take from hours to months or even many years. The metal whiskers are crystalline structures commonly having uniform cross sectional area along their entire length. Typical whisker dimensions are nominally on the order of only a few microns (um) across while their lengths can extend from a few microns to several millimeters. Metal whiskers pose a reliability hazard to electronic systems primarily as an electrical shorting hazard. The extremely narrow dimensions of metal whiskers can make observation with optical techniques very challenging. The videos herein were compiled to demonstrate the complexities associated with optical microscope inspection of electronic and mechanical components and assemblies for the presence or absence of metal whiskers. The importance of magnification, light source and angle of illumination play critical roles in being able to detect metal whiskers when present. Furthermore, it is demonstrated how improper techniques can easily obscure detection. It is hoped that these videos will improve the probability of detecting metal whiskers with optical inspection techniques.

  12. Precision 3-D microscopy with intensity modulated fibre optic scanners

    NASA Astrophysics Data System (ADS)

    Olmos, P.

    2016-01-01

    Optical 3-D imagers constitute a family of precision and useful instruments, easily available on the market in a wide variety of configurations and performances. However, besides their cost they usually provide an image of the object (i.e. a more or less faithful representation of the reality) instead of a truly object's reconstruction. Depending on the detailed working principles of the equipment, this reconstruction may become a challenging task. Here a very simple yet reliable device is described; it is able to form images of opaque objects by illuminating them with an optical fibre and collecting the reflected light with another fibre. Its 3-D capability comes from the spatial filtering imposed by the fibres together with their movement (scanning) along the three directions: transversal (surface) and vertical. This unsophisticated approach allows one to model accurately the entire optical process and to perform the desired reconstruction, finding that information about the surface which is of interest: its profile and its reflectance, ultimately related to the type of material.

  13. Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

    PubMed Central

    Masedunskas, Andrius; Porat-Shliom, Natalie; Tora, Muhibullah; Milberg, Oleg; Weigert, Roberto

    2013-01-01

    Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level. In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton. PMID:24022089

  14. Cornea microstructure and mechanical responses measured with nonlinear optical and optical coherence microscopy using sub-10-fs pulses

    PubMed Central

    Wu, Qiaofeng; Applegate, Brian E.; Yeh, Alvin T.

    2011-01-01

    A combined nonlinear optical microscopy (NLOM) and optical coherence microscopy (OCM) imaging system has been assembled in order to simultaneously capture co-registered volumetric images of corneal morphology and biochemistry. Tracking of cell nuclei visible in the OCM volume enabled the calculation of strain depth profile in response to changes in intraocular pressure for rabbit cornea stroma. Results revealed nonlinear responses with a depth dependent strain distribution, exhibiting smaller strains in the anterior and larger strains in the posterior stroma. Cross-sectional images of collagen lamellae, visible in NLOM, showed inhomogeneous collagen structure along the anterior-posterior direction that correlated well with the noted heterogeneous corneal mechanical responses. PMID:21559126

  15. In situ microscopy using adjustment-free optics

    NASA Astrophysics Data System (ADS)

    Suhr, Hajo; Herkommer, Alois M.

    2015-11-01

    In the past years, in situ microscopy has been demonstrated as a technique for monitoring the concentration and morphology of moving microparticles in agitated suspensions. However, up until now, this technique can only achieve a high resolution if a certain manual or automated effort is established for continuous precise focusing. Therefore, the application of in situ microscopes (ISMs) as sensors is inhibited in the cases where unattended operation is required. Here, we demonstrate a high-resolution ISM which, unlike others, is built as an entirely rigid construction, requiring no adjustments at all. This ISM is based on a specially designed water immersion objective with numerical aperture=0.75 and a working distance of 15 μm. The objective can be built exclusively from off-the-shelf parts and the front surface directly interfaces with the moving suspension. We show various applications of the system and demonstrate the imaging performance with submicron resolution within moving suspensions of microorganisms.

  16. Advanced Motion Compensation Methods for Intravital Optical Microscopy

    PubMed Central

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

    2013-01-01

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

  17. Time-domain optical coherence tomography with digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Massatsch, Pia; Charrière, Florian; Cuche, Etienne; Marquet, Pierre; Depeursinge, Christian D.

    2005-04-01

    We show that digital holography can be combined easily with optical coherence tomography approach. Varying the reference path length is the means used to acquire a series of holograms at different depths, providing after reconstruction images of slices at different depths in the specimen thanks to the short-coherence length of light source. A metallic object, covered by a 150-µm-thick onion cell, is imaged with high resolution. Applications in ophthalmology are shown: structures of the anterior eye, the cornea, and the iris, are studied on enucleated porcine eyes. Tomographic images of the iris border close to the pupil were obtained 165 µm underneath the eye surface.

  18. Three-dimensional reconstruction of paramecium primaurelia oral apparatus through confocal laser scanning optical microscopy

    NASA Astrophysics Data System (ADS)

    Beltrame, Francesco; Ramoino, Paola; Fato, Marco; Delmonte Corrado, Maria U.; Marcenaro, Giampiero; Crippa Franceschi, Tina

    1992-06-01

    Studies on the complementary mating types of Paramecium primaurelia (Protozoa, Ciliates) have shown that cell lines which differ from each other in mating type expression are characterized by different cell contents, organization, and physiology. Referring to these differences and to the differential rates of food vacuole formation, oral apparatuses of the two mating type cells are assumed to possibly differ from each other in some traits, such as, for instance, in their lengths. In our work, the highly organized oral structures are analyzed by means of a laser scanning confocal optical microscope (CLSM), which provides their 3-D visualization and measurement. The extraction of the 3-D intrinsic information related to the biological objects under investigation can be in turn related to their functional state, according to the classical paradigm of structure to function relationships identification. In our experiments, we acquired different data sets. These are optical slices of the biological sample under investigation, acquired in a confocal situation, through epi-illumination, in reflection, and, for comparison with conventional microscopy, 2-D images acquired via a standard TV camera coupled to the microscope itself. Our CLSM system is equipped with a laser beam at 488 and 514 nm and the data have been acquired with various steps of optical slicing, ranging from .04 to .25 micrometers. The volumes obtained by piling-up the slices are rendered through different techniques, some of them directly implemented on the workstation controlling the CLSM system, some of them on a SUN SPARC station 1, where the original data were transferred via an Ethernet link. In this last instance, original software has been developed for the visualization and animation of the 3-D structures, running under UNIX and X-Window, according to a ray-tracing algorithm.

  19. Nonparaxial vector-field modeling of optical coherence tomography and interferometric synthetic aperture microscopy.

    PubMed

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

    2007-09-01

    A large-aperture, electromagnetic model for coherent microscopy is presented and the inverse scattering problem is solved. Approximations to the model are developed for near-focus and far-from-focus operations. These approximations result in an image-reconstruction algorithm consistent with interferometric synthetic aperture microscopy (ISAM): this validates ISAM processing of optical-coherence-tomography and optical-coherence-microscopy data in a vectorial setting. Numerical simulations confirm that diffraction-limited resolution can be achieved outside the focal plane and that depth of focus is limited only by measurement noise and/or detector dynamic range. Furthermore, the model presented is suitable for the quantitative study of polarimetric coherent microscopy systems operating within the first Born approximation. PMID:17767224

  20. Combining microscopy with mesoscopy using optical and optoacoustic label-free modes

    NASA Astrophysics Data System (ADS)

    Soliman, Dominik; Tserevelakis, George J.; Omar, Murad; Ntziachristos, Vasilis

    2015-08-01

    Biology requires observations at multiple geometrical scales, a feature that is not typically offered by a single imaging modality. We developed a hybrid optical system that not only provides different contrast modes but also offers imaging at different geometrical scales, achieving uniquely broad resolution and a 1000-fold volume sampling increase compared to volumes scanned by optical microscopy. The system combines optoacoustic mesoscopy, optoacoustic microscopy and two-photon microscopy, the latter integrating second and third harmonic generation modes. Label-free imaging of a mouse ear and zebrafish larva ex-vivo demonstrates the contrast and scale complementarity provided by the hybrid system. We showcase the superior anatomical orientation offered by the label-free capacity and hybrid operation, over fluorescence microscopy, and the dynamic selection between field of view and resolution achieved, leading to new possibilities in biological visualization.

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

  2. Spatial compound imaging for fiber-bundle optic microscopy

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

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

  3. Characterization of a synthetic bioactive polymer by nonlinear optical microscopy

    PubMed Central

    Djaker, N.; Brustlein, S.; Rohman, G.; Huot, S.; de la Chapelle, M. Lamy; Migonney, V.

    2013-01-01

    Tissue Engineering is a new emerging field that offers many possibilities to produce three-dimensional and functional tissues like ligaments or scaffolds. The biocompatibility of these materials is crucial in tissue engineering, since they should be integrated in situ and should induce a good cell adhesion and proliferation. One of the most promising materials used for tissue engineering are polyesters such as Poly-ε-caprolactone (PCL), which is used in this work. In our case, the bio-integration is reached by grafting a bioactive polymer (pNaSS) on a PCL surface. Using nonlinear microscopy, PCL structure is visualized by SHG and proteins and cells by two-photon excitation autofluorescence generation. A comparative study between grafted and nongrafted polymer films is provided. We demonstrate that the polymer grafting improves the protein adsorption by a factor of 75% and increase the cell spreading onto the polymer surface. Since the spreading is directly related to cell adhesion and proliferation, we demonstrate that the pNaSS grafting promotes PCL biocompatibility. PMID:24466483

  4. Dynamics of solid lubrication as observed by optical microscopy

    NASA Technical Reports Server (NTRS)

    Sliney, H. E.

    1976-01-01

    A bench metallograph was converted into a 'micro contact imager' by the addition of a tribometer employing a steel ball in sliding contact with a glass disk. The sliding contact was viewed in real time by means of projection microscope optics. The dynamics of abrasive particles and of solid lubricant particles within the contact were observed in detail. The contact was characterized by a constantly changing pattern of elastic strain with the passage of surface discontinuities and solid particles. Abrasive particles fragmented upon entering the contact, embedded in one surface and scratched the other; in contrast, the solid lubricant particles flowed plastically into thin films. The rheological behavior of the lubricating solids gave every appearance of a paste-like consistency within the Hertzian contact.

  5. Dynamics of solid lubrication as observed by optical microscopy

    NASA Technical Reports Server (NTRS)

    Sliney, H. E.

    1976-01-01

    A bench metallograph was converted into a micro contact imager by the addition of a tribometer employing a steel ball in sliding contact with a glass disk. The sliding contact was viewed in real time by means of projection microscope optics. The dynamics of abrasive particles and of solid lubricant particles within the contact were observed in detail. The contact was characterized by a constantly changing pattern of elastic strain with the passage of surface discontinuities and solid particles. Abrasive particles fragmented upon entering the contact, embedded in one surface and scratched the other; in contrast, the solid lubricant particles flowed plastically into thin films. The rheological behavior of the lubricating solids gave every appearance of a paste-like consistency within the Hertzian contact.

  6. Neural imaging in songbirds using fiber optic fluorescence microscopy

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  7. Near-Field Scanning Optical Microscopy for High-Resolution Membrane Studies

    PubMed Central

    Huckabay, Heath A.; Armendariz, Kevin P.; Newhart, William H.; Wildgen, Sarah M.; Dunn, Robert C.

    2012-01-01

    The desire to directly probe biological structures on the length scales that they exist has driven the steady development of various high-resolution microscopy techniques. Among these, optical microscopy and, in particular, fluorescence-based approaches continue to occupy dominant roles in biological studies given their favorable attributes. Fluorescence microscopy is both sensitive and specific, is generally noninvasive toward biological samples, has excellent temporal resolution for dynamic studies, and is relatively inexpensive. Light-based microscopies can also exploit a myriad of contrast mechanisms based on spectroscopic signatures, energy transfer, polarization, and lifetimes to further enhance the specificity or information content of a measurement. Historically, however, spatial resolution has been limited to approximately half the wavelength due to the diffraction of light. Near-field scanning optical microscopy (NSOM) is one of several optical approaches currently being developed that combines the favorable attributes of fluorescence microscopy with superior spatial resolution. NSOM is particularly well suited for studies of both model and biological membranes and application to these systems is discussed. PMID:23086886

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

    PubMed

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

    2016-01-01

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

  9. Multi-color stimulated Raman scattering microscopy with a rapidly tunable optical parametric oscillator

    PubMed Central

    Kong, Lingjie; Ji, Minbiao; Holtom, Gary R.; Fu, Dan; Freudiger, Christian W.; Xie, X. Sunney

    2013-01-01

    Stimulated Raman scattering (SRS) microscopy allows label-free chemical imaging based on vibrational spectroscopy. Narrowband excitation with picosecond lasers creates the highest signal levels and enables imaging speeds up to video-rate, but sacrifices chemical specify in samples with overlapping bands compared to broadband (multiplex) excitation. We develope a rapidly tunable picosecond optical parametric oscillator with an electro-optical tunable Lyot filter, and demonstrate multi-color SRS microscopy with synchronized line-by-line wavelength tuning to avoid spectral artifacts due to sample movement. We show sensitive imaging of three different kinds of polymer beads and live HeLa cells with moving intracellular lipid droplets. PMID:23454943

  10. Optical Microscopy Characterization for Borehole U-15n#12 in Support of NCNS Source Physics Experiment

    SciTech Connect

    Wilson, Jennifer E.; Sussman, Aviva Joy

    2015-05-22

    Optical microscopy characterization of thin sections from corehole U-15n#12 is part of a larger material characterization effort for the Source Physics Experiment (SPE). The SPE program was conducted in Nevada with a series of explosive tests designed to study the generation and propagation of seismic waves inside Stock quartz monzonite. Optical microscopy analysis includes the following: 1) imaging of full thin sections (scans and mosaic maps); 2) high magnification imaging of petrographic texture (grain size, foliations, fractures, etc.); and 3) measurement of microfracture density.

  11. Chip-based optical microscopy for imaging membrane sieve plates of liver scavenger cells

    NASA Astrophysics Data System (ADS)

    Helle, Øystein I.; Øie, Cristina I.; McCourt, Peter; Ahluwalia, Balpreet S.

    2015-08-01

    The evanescent field on top of optical waveguides is used to image membrane network and sieve-plates of liver endothelial cells. In waveguide excitation, the evanescent field is dominant only near the surface (~100-150 nm) providing a default optical sectioning by illuminating fluorophores in close proximity to the surface and thus benefiting higher signal-to-noise ratio. The sieve plates of liver sinusoidal endothelial cells are present on the cell membrane, thus near-field waveguide chip-based microscopy configuration is preferred over epi-fluorescence. The waveguide chip is compatible with optical fiber components allowing easy multiplexing to different wavelengths. In this paper, we will discuss the challenges and opportunities provided by integrated optical microscopy for imaging cell membranes.

  12. Ultra-sensitive Magnetic Microscopy with an Optically Pumped Magnetometer

    NASA Astrophysics Data System (ADS)

    Kim, Young Jin; Savukov, Igor

    2016-04-01

    Optically pumped magnetometers (OPMs) based on lasers and alkali-metal vapor cells are currently the most sensitive non-cryogenic magnetic field sensors. Many applications in neuroscience and other fields require high-resolution, high-sensitivity magnetic microscopic measurements. In order to meet this demand we combined a cm-size spin-exchange relaxation-free (SERF) OPM and flux guides (FGs) to realize an ultra-sensitive FG-OPM magnetic microscope. The FGs serve to transmit the target magnetic flux to the OPM thus improving both the resolution and sensitivity to small magnetic objects. We investigated the performance of the FG-OPM device using experimental and numerical methods, and demonstrated that an optimized device can achieve a unique combination of high resolution (80 μm) and high sensitivity (8.1 pT/). In addition, we also performed numerical calculations of the magnetic field distribution in the FGs to estimate the magnetic noise originating from the domain fluctuations in the material of the FGs. We anticipate many applications of the FG-OPM device such as the detection of micro-biological magnetic fields; the detection of magnetic nano-particles; and non-destructive testing. From our theoretical estimate, an FG-OPM could detect the magnetic field of a single neuron, which would be an important milestone in neuroscience.

  13. Near-Field Optical Microscopy in the Infrared Range

    NASA Astrophysics Data System (ADS)

    de Wilde, Yannick; Lemoine, Paul-Arthur; Babuty, Arthur

    The infrared covers the region of the electromagnetic spectrum with wave numbers (optical frequencies) in the range 13 000 to 10 cm-1, which corresponds to the wavelength range from 780 nm to 1 mm. Infrared frequencies, in particular those in the mid-infrared (2 < λ < 25 μm), are especially attractive for probing materials, for they provide a way of identifying and quantifying their composition and structure, or probing their electronic and thermal properties. The absorption of infrared photons excites molecular vibrations and phonons. It is maximal at those frequencies associated with the vibrational modes, which depend on the kind of molecules and functional groups involved in the vibrations, or the crystal structure. So the frequencies of the absorption maxima form a spectral fingerprint of the material. Other factors such as the electron density and interactions between electrons, in semiconductors, metals, and superconductors, affect the dielectric properties at infrared frequencies, and are studied in the field of condensed matter. Regarding thermal imaging, infrared thermography is used to observe the temperature distribution at the surface of materials. The intensity of the thermal radiation emitted by a material depends on the temperature according to Planck's law. The temperature of a material can be determined by measuring the intensity of the infrared radiation it emits, once its emissivity is known.

  14. Ultra-sensitive magnetic microscopy with an optically pumped magnetometer

    DOE PAGESBeta

    Kim, Young Jin; Savukov, Igor Mykhaylovich

    2016-04-22

    Optically pumped magnetometers (OPMs) based on lasers and alkali-metal vapor cells are currently the most sensitive non-cryogenic magnetic field sensors. Many applications in neuroscience and other fields require high-resolution, high-sensitivity magnetic microscopic measurements. In order to meet this demand we combined a cm-size spin-exchange relaxation-free (SERF) OPM and flux guides (FGs) to realize an ultra-sensitive FG-OPM magnetic microscope. The FGs serve to transmit the target magnetic flux to the OPM thus improving both the resolution and sensitivity to small magnetic objects. We investigated the performance of the FG-OPM device using experimental and numerical methods, and demonstrated that an optimized devicemore » can achieve a unique combination of high resolution (80 μm) and high sensitivity (8.1 pT/). Additionally, we also performed numerical calculations of the magnetic field distribution in the FGs to estimate the magnetic noise originating from the domain fluctuations in the material of the FGs. We anticipate many applications of the FG-OPM device such as the detection of micro-biological magnetic fields; the detection of magnetic nano-particles; and non-destructive testing. From our theoretical estimate, an FG-OPM could detect the magnetic field of a single neuron, which would be an important milestone in neuroscience.« less

  15. Ultra-sensitive Magnetic Microscopy with an Optically Pumped Magnetometer.

    PubMed

    Kim, Young Jin; Savukov, Igor

    2016-01-01

    Optically pumped magnetometers (OPMs) based on lasers and alkali-metal vapor cells are currently the most sensitive non-cryogenic magnetic field sensors. Many applications in neuroscience and other fields require high-resolution, high-sensitivity magnetic microscopic measurements. In order to meet this demand we combined a cm-size spin-exchange relaxation-free (SERF) OPM and flux guides (FGs) to realize an ultra-sensitive FG-OPM magnetic microscope. The FGs serve to transmit the target magnetic flux to the OPM thus improving both the resolution and sensitivity to small magnetic objects. We investigated the performance of the FG-OPM device using experimental and numerical methods, and demonstrated that an optimized device can achieve a unique combination of high resolution (80 μm) and high sensitivity (8.1 pT/). In addition, we also performed numerical calculations of the magnetic field distribution in the FGs to estimate the magnetic noise originating from the domain fluctuations in the material of the FGs. We anticipate many applications of the FG-OPM device such as the detection of micro-biological magnetic fields; the detection of magnetic nano-particles; and non-destructive testing. From our theoretical estimate, an FG-OPM could detect the magnetic field of a single neuron, which would be an important milestone in neuroscience. PMID:27103463

  16. Ultra-sensitive Magnetic Microscopy with an Optically Pumped Magnetometer

    PubMed Central

    Kim, Young Jin; Savukov, Igor

    2016-01-01

    Optically pumped magnetometers (OPMs) based on lasers and alkali-metal vapor cells are currently the most sensitive non-cryogenic magnetic field sensors. Many applications in neuroscience and other fields require high-resolution, high-sensitivity magnetic microscopic measurements. In order to meet this demand we combined a cm-size spin-exchange relaxation-free (SERF) OPM and flux guides (FGs) to realize an ultra-sensitive FG-OPM magnetic microscope. The FGs serve to transmit the target magnetic flux to the OPM thus improving both the resolution and sensitivity to small magnetic objects. We investigated the performance of the FG-OPM device using experimental and numerical methods, and demonstrated that an optimized device can achieve a unique combination of high resolution (80 μm) and high sensitivity (8.1 pT/). In addition, we also performed numerical calculations of the magnetic field distribution in the FGs to estimate the magnetic noise originating from the domain fluctuations in the material of the FGs. We anticipate many applications of the FG-OPM device such as the detection of micro-biological magnetic fields; the detection of magnetic nano-particles; and non-destructive testing. From our theoretical estimate, an FG-OPM could detect the magnetic field of a single neuron, which would be an important milestone in neuroscience. PMID:27103463

  17. Generalized spectral method for near-field optical microscopy

    NASA Astrophysics Data System (ADS)

    Jiang, B.-Y.; Zhang, L. M.; Castro Neto, A. H.; Basov, D. N.; Fogler, M. M.

    2016-02-01

    Electromagnetic interaction between a sub-wavelength particle (the "probe") and a material surface (the "sample") is studied theoretically. The interaction is shown to be governed by a series of resonances corresponding to surface polariton modes localized near the probe. The resonance parameters depend on the dielectric function and geometry of the probe as well as on the surface reflectivity of the material. Calculation of such resonances is carried out for several types of axisymmetric probes: spherical, spheroidal, and pear-shaped. For spheroids, an efficient numerical method is developed, capable of handling cases of large or strongly momentum-dependent surface reflectivity. Application of the method to highly resonant materials, such as aluminum oxide (by itself or covered with graphene), reveals a rich structure of multi-peak spectra and nonmonotonic approach curves, i.e., the probe-sample distance dependence. These features also strongly depend on the probe shape and optical constants of the model. For less resonant materials such as silicon oxide, the dependence is weak, so that the spheroidal model is reliable. The calculations are done within the quasistatic approximation with radiative damping included perturbatively.

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

    PubMed

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

    2015-03-24

    We demonstrate multicolor three-dimensional super-resolution imaging with quantum dots (QSTORM). By combining quantum dot asynchronous spectral blueing with stochastic optical reconstruction microscopy and adaptive optics, we achieve three-dimensional imaging with 24 nm lateral and 37 nm axial resolution. By pairing two short-pass filters with two appropriate quantum dots, we are able to image single blueing quantum dots on two channels simultaneously, enabling multicolor imaging with high photon counts. PMID:25703291

  19. 3D defect detection using optical wide-field microscopy

    NASA Astrophysics Data System (ADS)

    Tympel, Volker; Schaaf, Marko; Srocka, Bernd

    2007-06-01

    We report a method to detect signed differences in two similar data sets representing 3-dimensional intensity profiles recorded by optical wide-field microscopes. The signed differences describe missing or unexpected intensity values, defined as defects. In technical applications like wafer and mask inspection, data sets often represent surfaces. The reported method is able to describe the size and position especially in relation to the neighboring surface and is called Three-Dimension-Aberration (TDA)-Technology. To increase the tool performance and to handle different sizes of defects a scaled bottom-up method is implemented and started with high reduced data sets for the search of large defects. Each analysis contains three steps. The first step is a correlation to calculate the displacement vector between the similar data sets. In the second step a new data set is created. The new data set consists of intensity differences. Extreme values in the data set represent the position of defects. By the use of linear and non-linear filters the stability of detection can be improved. If all differences are below a threshold the bottom-up method starts with the next larger scaled data set. In the other case it is assumed that the defect is detected and step three starts with the detection of the convex hull of the defect and the search of the neighboring surface. As a result the defect is described by a parameter set including the relative position. Because of the layered structure of the data set and the bottom-up technique the method is suitable for multi-core processor architectures.

  20. Harmonic demodulation and minimum enhancement factors in field-enhanced near-field optical microscopy.

    PubMed

    Scarpettini, A F; Bragas, A V

    2015-01-01

    Field-enhanced scanning optical microscopy relies on the design and fabrication of plasmonic probes which had to provide optical and chemical contrast at the nanoscale. In order to do so, the scattering containing the near-field information recorded in a field-enhanced scanning optical microscopy experiment, has to surpass the background light, always present due to multiple interferences between the macroscopic probe and sample. In this work, we show that when the probe-sample distance is modulated with very low amplitude, the higher the harmonic demodulation is, the better the ratio between the near-field signal and the interferometric background results. The choice of working at a given n harmonic is dictated by the experiment when the signal at the n + 1 harmonic goes below the experimental noise. We demonstrate that the optical contrast comes from the nth derivative of the near-field scattering, amplified by the interferometric background. By modelling the far and near field we calculate the probe-sample approach curves, which fit very well the experimental ones. After taking a great amount of experimental data for different probes and samples, we conclude with a table of the minimum enhancement factors needed to have optical contrast with field-enhanced scanning optical microscopy. PMID:25231792

  1. Dynamic structure and protein expression of the live embryonic heart captured by 2-photon light sheet microscopy and retrospective registration

    PubMed Central

    Trivedi, Vikas; Truong, Thai V.; Trinh, Le A.; Holland, Daniel B.; Liebling, Michael; Fraser, Scott E.

    2015-01-01

    We present an imaging and image reconstruction pipeline that captures the dynamic three-dimensional beating motion of the live embryonic zebrafish heart at subcellular resolution. Live, intact zebrafish embryos were imaged using 2-photon light sheet microscopy, which offers deep and fast imaging at 70 frames per second, and the individual optical sections were assembled into a full 4D reconstruction of the beating heart using an optimized retrospective image registration algorithm. This imaging and reconstruction platform permitted us to visualize protein expression patterns at endogenous concentrations in zebrafish gene trap lines. PMID:26114028

  2. Optical heterodyne-detected Raman-induced Kerr effect (OHD-RIKE) microscopy.

    PubMed

    Freudiger, Christian W; Roeffaers, Maarten B J; Zhang, Xu; Saar, Brian G; Min, Wei; Xie, X Sunney

    2011-05-12

    Label-free microscopy based on Raman scattering has been increasingly used in biomedical research to image samples that cannot be labeled or stained. Stimulated Raman scattering (SRS) microscopy allows signal amplification of the weak Raman signal for fast imaging speeds without introducing the nonresonant background and coherent image artifacts that are present in coherent anti-Stokes Raman scattering (CARS) microscopy. Here we present the Raman-induced Kerr effect (RIKE) as a contrast for label-free microscopy. RIKE allows us to measure different elements of the nonlinear susceptibility tensor, both the real and imaginary parts, by optical heterodyne detection (OHD-RIKE). OHD-RIKE microscopy provides information similar to polarization CARS (P-CARS) and interferometric CARS (I-CARS) microscopy, with a simple modification of the two-beam SRS microscopy setup. We show that, while OHD-RIKE microspectroscopy can be in principle more sensitive than SRS, it does not supersede SRS microscopy of heterogeneous biological samples, such as mouse skin tissue, because it is complicated by variations of linear birefringence across the sample. PMID:21504149

  3. Optical heterodyne-detected Raman-Induced Kerr Effect (OHD-RIKE) microscopy

    PubMed Central

    Freudiger, Christian W.; Roeffaers, Maarten B. J.; Zhang, Xu; Saar, Brian G.; Min, X., Wei; Xie, Sunney

    2012-01-01

    Label-free microscopy based on Raman scattering has been increasingly used in biomedical research to image samples that cannot be labeled or stained. Stimulated Raman scattering (SRS) microscopy, allows signal amplification of the weak Raman signal for fast imaging speeds without introducing the non-resonant background and coherent image artifacts that are present in coherent anti-Stokes Raman scattering (CARS) microscopy. Here we present the Raman-induced Kerr effect (RIKE) as a contrast for label-free microscopy. RIKE allows us to measure different elements of the non-linear susceptibility tensor, both the real and imaginary parts by optical heterodyne detection (OHD-RIKE). OHD-RIKE microscopy provides information similar to polarization CARS (P-CARS) and interferometric CARS (I-CARS) microscopy, with a simple modification of the two-beam SRS microscopy setup. We show that while OHD-RIKE micro-spectroscopy can be in principle more sensitive than SRS, it does not supersede SRS microscopy of heterogeneous biological samples, such as mouse skin tissue, because it is complicated by variations of linear birefringence across the sample. PMID:21504149

  4. Combined lineage mapping and gene expression profiling of embryonic brain patterning using ultrashort pulse microscopy and image registration

    NASA Astrophysics Data System (ADS)

    Gibbs, Holly C.; Dodson, Colin R.; Bai, Yuqiang; Lekven, Arne C.; Yeh, Alvin T.

    2014-12-01

    During embryogenesis, presumptive brain compartments are patterned by dynamic networks of gene expression. The spatiotemporal dynamics of these networks, however, have not been characterized with sufficient resolution for us to understand the regulatory logic resulting in morphogenetic cellular behaviors that give the brain its shape. We have developed a new, integrated approach using ultrashort pulse microscopy [a high-resolution, two-photon fluorescence (2PF)-optical coherence microscopy (OCM) platform using 10-fs pulses] and image registration to study brain patterning and morphogenesis in zebrafish embryos. As a demonstration, we used time-lapse 2PF to capture midbrain-hindbrain boundary morphogenesis and a wnt1 lineage map from embryos during brain segmentation. We then performed in situ hybridization to deposit NBT/BCIP, where wnt1 remained actively expressed, and reimaged the embryos with combined 2PF-OCM. When we merged these datasets using morphological landmark registration, we found that the mechanism of boundary formation differs along the dorsoventral axis. Dorsally, boundary sharpening is dominated by changes in gene expression, while ventrally, sharpening may be accomplished by lineage sorting. We conclude that the integrated visualization of lineage reporter and gene expression domains simultaneously with brain morphology will be useful for understanding how changes in gene expression give rise to proper brain compartmentalization and structure.

  5. Noninvasive harmonics optical microscopy for long-term observation of embryonic nervous system development in vivo.

    PubMed

    Chen, Szu-Yu; Hsieh, Cho-Shuen; Chu, Shi-Wei; Lin, Cheng-Yung; Ko, Ching-Yi; Chen, Yi-Chung; Tsai, Huai-Jen; Hu, Chin-Hwa; Sun, Chi-Kuang

    2006-01-01

    Nervous system development is a complicated dynamic process, and many mechanisms remain unknown. By utilizing endogenous second-harmonic-generation as the contrast of polarized nerve fibers and third-harmonic-generation (THG) to reveal morphological changes, we have successfully observed the vertebrate embryonic nervous development from the very beginning based on a 1230-nm light source. The dynamic development of the nerve system within a live zebrafish embryo can be recorded continuously more than 20 hr without fluorescence markers. Since the THG process is not limited by the time of gene expression and differentiation as fluorescence-based techniques are, the observable stages can be advanced to the very beginning of the development process. The complete three-dimensional brain development from a neural plate to a neural tube can be uncovered with a submicron lateral resolution. We have, for the first time, also reported the generation of SHG from myelinated nerve fibers and the outer segment of the photoreceptors with a stacked membrane structure. Our study clearly indicates the fact that higher-harmonics-based optical microscopy has the strong potential to long-term in vivo study of the nervous system, including genetic disorders of the nervous system, axon pathfinding, neural regeneration, neural repair, and neural stem cell development. PMID:17092171

  6. Imaging of matrix-disorder in normal and pathological human dermis using nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Zhuo, Shuangmu; Chen, Jianxin; Xie, Shusen; Zheng, Liqin; Jiang, Xingshan

    2009-11-01

    In dermis, collagen and elastin are important structural proteins of extracellular maxtrix. The matrix-disorder is associated with various physiologic processes, such as localized scleroderma, anetoderma, photoaging. In this work, we demonstrate the capability of nonlinear optical microscopy in imaging structural proteins in normal and pathological human dermis.

  7. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

    PubMed Central

    Neuman, Keir C.; Nagy, Attila

    2012-01-01

    Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. These techniques are described and illustrated with examples highlighting current capabilities and limitations. PMID:18511917

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

    PubMed

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

    2015-01-01

    Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes. PMID:26625116

  9. Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy

    PubMed Central

    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. PMID:26625116

  10. Low coherence full field interference microscopy or optical coherence tomography: recent advances, limitations and future trends

    NASA Astrophysics Data System (ADS)

    Abdulhalim, I.

    2013-04-01

    Although low coherence microscopy (LCM) has been known for long time in the context of interference microscopy, coherence radar and white light interferometry, the whole subject has attracted a wide interest in the last two decades particularly accelerated by the entrance of OCT, as a noninvasive powerful technique for biomedical imaging. Today LCM can be classified into two types, both acts as three-dimensional imaging tool. The first is low temporal coherence microscopy; also known as optical coherence tomography (OCT), which is being used for medical diagnostics. The second is full field OCT in various modes and applied to various applications. FF-OCT uses low spatial and temporal coherence similar to the well-known coherence probe microscope (CPM) that have been in use for long time in optical metrology. The CPM has many advantages over conventional microscopy in its ability to discriminate between different transparent layers in a scattering medium thus allowing for precise noninvasive optical probing of dense tissue and other turbid media. In this paper the status of this technology in optical metrology applications will be discussed, on which we have been working to improve its performance, as well as its limitations and future prospective.

  11. DMD-based LED-illumination Super-resolution and optical sectioning microscopy

    PubMed Central

    Dan, Dan; Lei, Ming; Yao, Baoli; Wang, Wen; Winterhalder, Martin; Zumbusch, Andreas; Qi, Yujiao; Xia, Liang; Yan, Shaohui; Yang, Yanlong; Gao, Peng; Ye, Tong; Zhao, Wei

    2013-01-01

    Super-resolution three-dimensional (3D) optical microscopy has incomparable advantages over other high-resolution microscopic technologies, such as electron microscopy and atomic force microscopy, in the study of biological molecules, pathways and events in live cells and tissues. We present a novel approach of structured illumination microscopy (SIM) by using a digital micromirror device (DMD) for fringe projection and a low-coherence LED light for illumination. The lateral resolution of 90 nm and the optical sectioning depth of 120 μm were achieved. The maximum acquisition speed for 3D imaging in the optical sectioning mode was 1.6×107 pixels/second, which was mainly limited by the sensitivity and speed of the CCD camera. In contrast to other SIM techniques, the DMD-based LED-illumination SIM is cost-effective, ease of multi-wavelength switchable and speckle-noise-free. The 2D super-resolution and 3D optical sectioning modalities can be easily switched and applied to either fluorescent or non-fluorescent specimens. PMID:23346373

  12. Tuning Localized Surface Plasmon Resonance in Scanning Near-Field Optical Microscopy Probes.

    PubMed

    Vasconcelos, Thiago L; Archanjo, Bráulio S; Fragneaud, Benjamin; Oliveira, Bruno S; Riikonen, Juha; Li, Changfeng; Ribeiro, Douglas S; Rabelo, Cassiano; Rodrigues, Wagner N; Jorio, Ado; Achete, Carlos A; Cançado, Luiz Gustavo

    2015-06-23

    A reproducible route for tuning localized surface plasmon resonance in scattering type near-field optical microscopy probes is presented. The method is based on the production of a focused-ion-beam milled single groove near the apex of electrochemically etched gold tips. Electron energy-loss spectroscopy and scanning transmission electron microscopy are employed to obtain highly spatially and spectroscopically resolved maps of the milled probes, revealing localized surface plasmon resonance at visible and near-infrared wavelengths. By changing the distance L between the groove and the probe apex, the localized surface plasmon resonance energy can be fine-tuned at a desired absorption channel. Tip-enhanced Raman spectroscopy is applied as a test platform, and the results prove the reliability of the method to produce efficient scattering type near-field optical microscopy probes. PMID:26027751

  13. Adaptive optics in spinning disk microscopy: improved contrast and brightness by a simple and fast method.

    PubMed

    Fraisier, V; Clouvel, G; Jasaitis, A; Dimitrov, A; Piolot, T; Salamero, J

    2015-09-01

    Multiconfocal microscopy gives a good compromise between fast imaging and reasonable resolution. However, the low intensity of live fluorescent emitters is a major limitation to this technique. Aberrations induced by the optical setup, especially the mismatch of the refractive index and the biological sample itself, distort the point spread function and further reduce the amount of detected photons. Altogether, this leads to impaired image quality, preventing accurate analysis of molecular processes in biological samples and imaging deep in the sample. The amount of detected fluorescence can be improved with adaptive optics. Here, we used a compact adaptive optics module (adaptive optics box for sectioning optical microscopy), which was specifically designed for spinning disk confocal microscopy. The module overcomes undesired anomalies by correcting for most of the aberrations in confocal imaging. Existing aberration detection methods require prior illumination, which bleaches the sample. To avoid multiple exposures of the sample, we established an experimental model describing the depth dependence of major aberrations. This model allows us to correct for those aberrations when performing a z-stack, gradually increasing the amplitude of the correction with depth. It does not require illumination of the sample for aberration detection, thus minimizing photobleaching and phototoxicity. With this model, we improved both signal-to-background ratio and image contrast. Here, we present comparative studies on a variety of biological samples. PMID:25940062

  14. Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging

    PubMed Central

    Vakoc, Benjamin J; Lanning, Ryan M; Tyrrell, James A; Padera, Timothy P; Bartlett, Lisa A; Stylianopoulos, Triantafyllos; Munn, Lance L; Tearney, Guillermo J; Fukumura, Dai; Jain, Rakesh K; Bouma, Brett E

    2009-01-01

    Intravital multiphoton microscopy has provided powerful mechanistic insights into health and disease, and has become a common instrument in the modern biological laboratory. The requisite high numerical aperture and exogenous contrast agents that enable multiphoton microscopy, however, limit ability to investigate substantial tissue volumes or to probe dynamic changes repeatedly over prolonged periods. Here, we introduce optical frequency domain imaging (OFDI) as an intravital microscopy that circumvents the technical limitations of multiphoton microscopy and, as a result, provides unprecedented access to previously unexplored, critically important aspects of tissue biology. Using novel OFDI-based approaches and entirely intrinsic mechanisms of contrast, we present rapid and repeated measurements of tumor angiogenesis, lymphangiogenesis, tissue viability and both vascular and cellular responses to therapy, thereby demonstrating the potential of OFDI to facilitate the exploration of physiological and pathological processes and the evaluation of treatment strategies. PMID:19749772

  15. Comprehensive study of unexpected microscope condensers formed in sample arrangements commonly used in optical microscopy.

    PubMed

    Desai, Darshan B; Aldawsari, Mabkhoot Mudith S; Alharbi, Bandar Mohammed H; Sen, Sanchari; Grave de Peralta, Luis

    2015-09-01

    We show that various setups for optical microscopy which are commonly used in biomedical laboratories behave like efficient microscope condensers that are responsible for observed subwavelength resolution. We present a series of experiments and simulations that reveal how inclined illumination from such unexpected condensers occurs when the sample is perpendicularly illuminated by a microscope's built-in white-light source. In addition, we demonstrate an inexpensive add-on optical module that serves as an efficient and lightweight microscope condenser. Using such add-on optical module in combination with a low-numerical-aperture objective lens and Fourier plane imaging microscopy technique, we demonstrate detection of photonic crystals with a period nearly eight times smaller than the Rayleigh resolution limit. PMID:26368905

  16. Nonlinear-microscopy optical-pulse sources based on mode-locked semiconductor lasers.

    PubMed

    Yokoyama, H; Sato, A; Guo, H-C; Sato, K; Mure, M; Tsubokawa, H

    2008-10-27

    We developed picosecond optical-pulse sources suitable for multiphoton microscopy based on mode-locked semiconductor lasers. Using external-cavity geometry, stable hybrid mode locking was achieved at a repetition rate of 500 MHz. Semiconductor optical amplifiers driven by synchronized electric pulses reached subharmonic optical-pulse repetition rates of 1-100 MHz. Two-stage Yb-doped fiber amplifiers produced optical pulses of 2 ps duration, with a peak power of a few kilowatts at a repetition rate of 10 MHz. These were employed successfully for nonlinear-optic bio-imaging using two-photon fluorescence, second-harmonic generation, and sum-frequency generation of synchronized two-color pulses. PMID:18958056

  17. Recognition of serous ovarian tumors in human samples by multimodal nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Adur, Javier; Pelegati, Vitor B.; Costa, Leverson F. L.; Pietro, Luciana; de Thomaz, Andre A.; Almeida, Diogo B.; Bottcher-Luiz, Fatima; Andrade, Liliana A. L. A.; Cesar, Carlos L.

    2011-09-01

    We used a multimodal nonlinear optics microscopy, specifically two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG/THG) microscopies, to observe pathological conditions of ovarian tissues obtained from human samples. We show that strong TPEF + SHG + THG signals can be obtained in fixed samples stained with hematoxylin and eosin (H&E) stored for a very long time, and that H&E staining enhanced the THG signal. We then used the multimodal TPEF-SHG-THG microscopies in a stored file of H&E stained samples of human ovarian cancer to obtain complementary information about the epithelium/stromal interface, such as the transformation of epithelium surface (THG) and the overall fibrillary tissue architecture (SHG). This multicontrast nonlinear optics microscopy is able to not only differentiate between cancerous and healthy tissue, but can also distinguish between normal, benign, borderline, and malignant specimens according to their collagen disposition and compression levels within the extracellular matrix. The dimensions of the layers of epithelia can also be measured precisely and automatically. Our data demonstrate that optical techniques can detect pathological changes associated with ovarian cancer.

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

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  20. Multifunctional imaging of endogenous contrast by simultaneous nonlinear and optical coherence microscopy of thick tissues.

    PubMed

    Yazdanfar, Siavash; Chen, Yen Yu; So, Peter T C; Laiho, Lily H

    2007-07-01

    A variety of high resolution optical microscopy techniques have been developed in recent years for basic and clinical studies of biological systems. We demonstrate a trimodal microscope combining optical coherence microscopy (OCM) with two forms of nonlinear microscopy, namely two-photon excited fluorescence (2PF) and second harmonic generation (SHG), for imaging turbid media. OCM combines the advantages of confocal detection and coherence gating for structural imaging in highly scattering tissues. Nonlinear microscopy enables the detection of biochemical species, such as elastin, NAD(P)H, and collagen. While 2PF arises from nonlinear excitation of fluorescent species, SHG is a form of nonlinear scattering observed in materials that lack a center of inversion symmetry, such as type I collagen. Characterization of the microscope showed nearly diffraction-limited spatial resolution in all modalities. Images were obtained in fish scales and excised human skin samples. The primary endogenous sources of contrast in the dermis were due to elastin autofluorescence and collagen SHG. Multimodal microscopy allows the simultaneous visualization of structural and functional information of biological systems. PMID:17323366

  1. Probing ultrafast spin dynamics with optical pump-probe scanning tunnelling microscopy.

    PubMed

    Yoshida, Shoji; Aizawa, Yuta; Wang, Zi-han; Oshima, Ryuji; Mera, Yutaka; Matsuyama, Eiji; Oigawa, Haruhiro; Takeuchi, Osamu; Shigekawa, Hidemi

    2014-08-01

    Studies of spin dynamics in low-dimensional systems are important from both fundamental and practical points of view. Spin-polarized scanning tunnelling microscopy allows localized spin dynamics to be characterized and plays important roles in nanoscale science and technology. However, nanoscale analysis of the ultrafast dynamics of itinerant magnetism, as well as its localized characteristics, should be pursued to advance further the investigation of quantum dynamics in functional structures of small systems. Here, we demonstrate the optical pump-probe scanning tunnelling microscopy technique, which enables the nanoscale probing of spin dynamics with the temporal resolution corresponding, in principle, to the optical pulse width. Spins are optically oriented using circularly polarized light, and their dynamics are probed by scanning tunnelling microscopy based on the optical pump-probe method. Spin relaxation in a single quantum well with a width of 6 nm was observed with a spatial resolution of ∼ 1 nm. In addition to spin relaxation dynamics, spin precession, which provides an estimation of the Landé g factor, was observed successfully. PMID:24974938

  2. Probing ultrafast spin dynamics with optical pump-probe scanning tunnelling microscopy

    NASA Astrophysics Data System (ADS)

    Yoshida, Shoji; Aizawa, Yuta; Wang, Zi-Han; Oshima, Ryuji; Mera, Yutaka; Matsuyama, Eiji; Oigawa, Haruhiro; Takeuchi, Osamu; Shigekawa, Hidemi

    2014-08-01

    Studies of spin dynamics in low-dimensional systems are important from both fundamental and practical points of view. Spin-polarized scanning tunnelling microscopy allows localized spin dynamics to be characterized and plays important roles in nanoscale science and technology. However, nanoscale analysis of the ultrafast dynamics of itinerant magnetism, as well as its localized characteristics, should be pursued to advance further the investigation of quantum dynamics in functional structures of small systems. Here, we demonstrate the optical pump-probe scanning tunnelling microscopy technique, which enables the nanoscale probing of spin dynamics with the temporal resolution corresponding, in principle, to the optical pulse width. Spins are optically oriented using circularly polarized light, and their dynamics are probed by scanning tunnelling microscopy based on the optical pump-probe method. Spin relaxation in a single quantum well with a width of 6 nm was observed with a spatial resolution of ~1 nm. In addition to spin relaxation dynamics, spin precession, which provides an estimation of the Landé g factor, was observed successfully.

  3. Nonlinear optical microscopy for label-free detection of gastrointestinal neuroendocrine tumors.

    PubMed

    Li, Lianhuang; Jiang, Liwei; Chen, Zhifen; Kang, Deyong; Yang, Zhenrong; Liu, Xing; Jiang, Weizhong; Zhuo, Shuangmu; Guan, Guoxian; Zhou, Yongjian; Chen, Jianxin

    2016-09-01

    Neuroendocrine tumors (NETs), which are rare and slow-growing neoplasms, pose a diagnostic challenge as they are clinically silent at the time of presentation. Here, gastrointestinal neuroendocrine tumors were researched by nonlinear microscopy, and results demonstrate that this technique has the capability to identify neuroendocrine tumors in the absence of labels and can, in particular, detect rare neuroendocrine tumor cells, vascular invasion, desmoplastic reaction, and fibroelastosis induced by neuroendocrine tumors. These conclusions highlight the possibility of nonlinear optical microscopy as a diagnostic tool for label-freely differentiating neuroendocrine tumors by these histopathologic features. PMID:27299572

  4. Two-Photon Microscopy with Diffractive Optical Elements and Spatial Light Modulators

    PubMed Central

    Watson, Brendon O.; Nikolenko, Volodymyr; Araya, Roberto; Peterka, Darcy S.; Woodruff, Alan; Yuste, Rafael

    2010-01-01

    Two-photon microscopy is often performed at slow frame rates due to the need to serially scan all points in a field of view with a single laser beam. To overcome this problem, we have developed two optical methods that split and multiplex a laser beam across the sample. In the first method a diffractive optical element (DOE) generates a fixed number of beamlets that are scanned in parallel resulting in a corresponding increase in speed or in signal-to-noise ratio in time-lapse measurements. The second method uses a computer-controlled spatial light modulator (SLM) to generate any arbitrary spatio-temporal light pattern. With an SLM one can image or photostimulate any predefined region of the image such as neurons or dendritic spines. In addition, SLMs can be used to mimic a large number of optical transfer functions including light path corrections as adaptive optics. PMID:20859526

  5. Ultrasound-modulated optical microscopy for ex-vivo imaging of scattering biological tissue

    NASA Astrophysics Data System (ADS)

    Kothapalli, Sri-Rajasekhar; Wang, Lihong V.

    2009-02-01

    Ultrasound-modulated optical microscopy (UOM) based on a long-cavity confocal Fabry-Perot interferometer (CFPI) [J.Biomed.Opt. 13(5), 0504046, (2008)] is used for real time detection of multiply scattered light modulated by high frequency (30 MHz) ultrasound pulses propagating in an optically strongly scattering medium. In this article, we use this microscope to study the dependence of ultrasound-modulated optical signals on the optical absorption of objects embedded about 3 mm deep in tissue mimicking phantoms. These results demonstrate that the dependence is nearly linear. Most importantly, we imaged blood vasculature and melanin in highly scattering tissue samples from a mouse and a rat. Thus UOM can be used to study the morphology of blood vasculature and blood-associated functional parameters, such as oxygen saturation.

  6. Doppler optical coherence microscopy and tomography applied to inner ear mechanics

    SciTech Connect

    Page, Scott; Freeman, Dennis M.; Ghaffari, Roozbeh

    2015-12-31

    While it is clear that cochlear traveling waves underlie the extraordinary sensitivity, frequency selectivity, and dynamic range of mammalian hearing, the underlying micromechanical mechanisms remain unresolved. Recent advances in low coherence measurement techniques show promise over traditional laser Doppler vibrometry and video microscopy, which are limited by low reflectivities of cochlear structures and restricted optical access. Doppler optical coherence tomography (DOCT) and Doppler optical coherence microscopy (DOCM) both utilize a broadband source to limit constructive interference of scattered light to a small axial depth called a coherence gate. The coherence gate can be swept axially to image and measure sub-nanometer motions of cochlear structures throughout the cochlear partition. The coherence gate of DOCT is generally narrower than the confocal gate of the focusing optics, enabling increased axial resolution (typically 15 μm) within optical sections of the cochlear partition. DOCM, frequently implemented in the time domain, centers the coherence gate on the focal plane, achieving enhanced lateral and axial resolution when the confocal gate is narrower than the coherence gate. We compare these two complementary systems and demonstrate their utility in studying cellular and micromechanical mechanisms involved in mammalian hearing.

  7. Structured light optical microscopy for three-dimensional reconstruction of technical surfaces

    NASA Astrophysics Data System (ADS)

    Kettel, Johannes; Reinecke, Holger; Müller, Claas

    2016-04-01

    In microsystems technology quality control of micro structured surfaces with different surface properties is playing an ever more important role. The process of quality control incorporates three-dimensional (3D) reconstruction of specularand diffusive reflecting technical surfaces. Due to the demand on high measurement accuracy and data acquisition rates, structured light optical microscopy has become a valuable solution to solve this problem providing high vertical and lateral resolution. However, 3D reconstruction of specular reflecting technical surfaces still remains a challenge to optical measurement principles. In this paper we present a measurement principle based on structured light optical microscopy which enables 3D reconstruction of specular- and diffusive reflecting technical surfaces. It is realized using two light paths of a stereo microscope equipped with different magnification levels. The right optical path of the stereo microscope is used to project structured light onto the object surface. The left optical path is used to capture the structured illuminated object surface with a camera. Structured light patterns are generated by a Digital Light Processing (DLP) device in combination with a high power Light Emitting Diode (LED). Structured light patterns are realized as a matrix of discrete light spots to illuminate defined areas on the object surface. The introduced measurement principle is based on multiple and parallel processed point measurements. Analysis of the measured Point Spread Function (PSF) by pattern recognition and model fitting algorithms enables the precise calculation of 3D coordinates. Using exemplary technical surfaces we demonstrate the successful application of our measurement principle.

  8. Doppler optical coherence microscopy and tomography applied to inner ear mechanics

    NASA Astrophysics Data System (ADS)

    Page, Scott; Ghaffari, Roozbeh; Freeman, Dennis M.

    2015-12-01

    While it is clear that cochlear traveling waves underlie the extraordinary sensitivity, frequency selectivity, and dynamic range of mammalian hearing, the underlying micromechanical mechanisms remain unresolved. Recent advances in low coherence measurement techniques show promise over traditional laser Doppler vibrometry and video microscopy, which are limited by low reflectivities of cochlear structures and restricted optical access. Doppler optical coherence tomography (DOCT) and Doppler optical coherence microscopy (DOCM) both utilize a broadband source to limit constructive interference of scattered light to a small axial depth called a coherence gate. The coherence gate can be swept axially to image and measure sub-nanometer motions of cochlear structures throughout the cochlear partition. The coherence gate of DOCT is generally narrower than the confocal gate of the focusing optics, enabling increased axial resolution (typically 15 μm) within optical sections of the cochlear partition. DOCM, frequently implemented in the time domain, centers the coherence gate on the focal plane, achieving enhanced lateral and axial resolution when the confocal gate is narrower than the coherence gate. We compare these two complementary systems and demonstrate their utility in studying cellular and micromechanical mechanisms involved in mammalian hearing.

  9. Absorption Coefficient Imaging by Near-Field Scanning Optical Microscopy in Bacteria

    NASA Astrophysics Data System (ADS)

    de Paula, Ana M.; Chaves, Claudilene R.; Silva, Haroldo B.; Weber, Gerald

    2003-06-01

    We present a method for obtaining a position-dependent absorption coefficient from near-field scanning optical transmission microscopy. We show that the optical transmission intensity can be combined with the topography, resulting into an absorption coefficient that simplifies the analysis of different materials within a sample. The method is tested with the dye rhodamine 6G, and we show some analysis in biological samples such as bacteria Klebsiella pneumoniae and Pseudomonas aeruginosa . The calculated absorption coefficient images show important details of the bacteria, in particular for P. aeruginosa , in which membrane vesicles are clearly seen.

  10. Critical dimension metrology by through-focus scanning optical microscopy beyond the 22 nm node

    NASA Astrophysics Data System (ADS)

    Attota, Ravikiran; Bunday, Benjamin; Vartanian, Victor

    2013-06-01

    We present results using simulations and experiments to demonstrate metrological applications of the through-focus scanning optical microscopy (TSOM) down to features at and well below the International Technology Roadmap for Semiconductors' 22 nm node. The TSOM method shows the ability to detect sub-nanometer, three-dimensional shape variations such as line height, sidewall angle, width, and pitch in fins of fin-shaped field effect transistor structures using conventional optical microscopes. In addition, the method requires targets substantially smaller than the conventional target size. These results provide insight into the applicability of TSOM for economical critical dimension and yield enhancement metrology.

  11. Monitoring cells in engineered tissues with optical coherence phase microscopy: Optical phase fluctuations as endogenous sources of contrast

    NASA Astrophysics Data System (ADS)

    Bagnaninchi, P. O.; Holmes, Christina; Tabrizian, Maryam

    2013-02-01

    There is a need in tissue engineering to monitor cell growth and health within 3D constructs non-invasively and in a label-free manner. We have previously shown that optical coherence phase microscopy was sensitive enough to monitor intracellular motion. Here we demonstrate that intracellular motility can be used as an endogeneous contrast agent to image cells in various 3D engineered tissue architectures. Phase and intensity-based reconstruction algorithms are compared. In this study, we used an optical coherence phase microscope set up in a common path configuration, developed around a Callisto OCT engine (Thorlbas) centred at 930nm and an inverted microscope with a custom scanning head. Intensity data were used to perform in-depth microstructural imaging. In addition, phase fluctuations were measured by collecting several successive B scans at the same location, and the first time derivative of the phase, i.e. time fluctuations, was analysed over the acquisition time interval to map the motility. Alternative intensity-based Doppler variance algorithms were also investigated. Two distinct scaffold systems seeded with adult stem cells; algimatrix (Invitrogen) and custom microfabricated poly(D,L-lactic-co-glycolic acid) fibrous scaffolds, as well as cell pellets were imaged. We showed that optical phase fluctuations resulting from intracellular motility can be used as an endogenous source of contrast for optical coherence phase microscopy enabling the distinction of viable cells from the surrounding scaffold.

  12. Vertically integrated optics for ballistic electron emission luminescence: Device and microscopy characterizations

    NASA Astrophysics Data System (ADS)

    Yi, Wei; Appelbaum, Ian; Russell, Kasey J.; Narayanamurti, Venkatesh; Schalek, Richard; Hanson, Micah P.; Gossard, Arthur C.

    2006-07-01

    By integrating a p-i-n photodiode photodetector directly into a ballistic electron emission luminescence (BEEL) heterostructure with GaAs quantum-well active region, we have obtained a photon detection efficiency of more than 10%. This is many orders of magnitude higher than conventional far-field detection scheme with the most sensitive single-photon counters, enabling BEEL microscopy in systems with no optical components. Detailed analysis shows found a parasitic bipolar injection in parallel with the desired optical coupling between the BEEL heterostructure and the integrated photodiode beyond a characteristic collector bias, which may be solved by improved device design or limiting the operating window of the collector bias. Preliminary BEEL microscopy images of a homogeneous GaAs quantum-well luminescent layer show lateral variations of photon emission correlated with the collector current injection level modulated by surface features or interface defects.

  13. New method for fast morphological characterization of organic polycrystalline films by polarized optical microscopy

    NASA Astrophysics Data System (ADS)

    He, Xiao-Chuan; Yang, Jian-Bing; Yan, Dong-Hang; Weng, Yu-Xiang

    2015-07-01

    A new method to visualize the large-scale crystal grain morphology of organic polycrystalline films is proposed. First, optical anisotropic transmittance images of polycrystalline zinc phthalocyanine (ZnPc) films vacuum deposited by weak epitaxial growth (WEG) method were acquired with polarized optical microscopy (POM). Then morphology properties including crystal grain size, distribution, relative orientation, and crystallinity were derived from these images by fitting with a transition dipole model. At last, atomic force microscopy (AFM) imaging was carried out to confirm the fitting and serve as absolute references. This method can be readily generalized to other organic polycrystalline films, thus providing an efficient way to access the large-scale morphologic properties of organic polycrystalline films, which may prove to be useful in industry as a film quality monitoring method. Project supported by the National Natural Science Foundation of China (Grant No. 20933010) and the National Basic Research Program of China (Grant No. 2013CB834800).

  14. Intravital imaging of amyloid plaques in a transgenic mouse model using optical-resolution photoacoustic microscopy

    PubMed Central

    Hu, Song; Yan, Ping; Maslov, Konstantin; Lee, Jin-Moo; Wang, Lihong V.

    2010-01-01

    We report optical-resolution photoacoustic microscopy (OR-PAM) for in vivo imaging of amyloid plaques in an Alzheimer’s disease mouse model. Validation using conventional fluorescence microscopy and multiphoton microscopy shows that OR-PAM has sufficient sensitivity and spatial resolution to identify amyloid plaques in living brains. In addition, with dual-wavelength OR-PAM, the three-dimensional morphology of amyloid plaques and the surrounding microvasculature are imaged simultaneously through a cranial window without angiographic contrast agents. OR-PAM, capable of providing both exogenous molecular contrast and endogenous hemoglobin contrast, has the potential to serve as a new technology for in vivo microscopic observations of cerebral plaque deposits. PMID:20016651

  15. Nano-scale measurement of biomolecules by optical microscopy and semiconductor nanoparticles

    PubMed Central

    Ichimura, Taro; Jin, Takashi; Fujita, Hideaki; Higuchi, Hideo; Watanabe, Tomonobu M.

    2014-01-01

    Over the past decade, great developments in optical microscopy have made this technology increasingly compatible with biological studies. Fluorescence microscopy has especially contributed to investigating the dynamic behaviors of live specimens and can now resolve objects with nanometer precision and resolution due to super-resolution imaging. Additionally, single particle tracking provides information on the dynamics of individual proteins at the nanometer scale both in vitro and in cells. Complementing advances in microscopy technologies has been the development of fluorescent probes. The quantum dot, a semi-conductor fluorescent nanoparticle, is particularly suitable for single particle tracking and super-resolution imaging. This article overviews the principles of single particle tracking and super resolution along with describing their application to the nanometer measurement/observation of biological systems when combined with quantum dot technologies. PMID:25120488

  16. High Resolution Phase-Sensitive Magnetomotive Optical Coherence Microscopy for Tracking Magnetic Microbeads and Cellular Mechanics

    PubMed Central

    Crecea, Vasilica; Graf, Benedikt W.; Kim, Taewoo; Popescu, Gabriel; Boppart, Stephen A.

    2014-01-01

    We present a real-time multimodal near-infrared imaging technology that tracks externally induced axial motion of magnetic microbeads in single cells in culture. The integrated multimodal imaging technique consists of phase-sensitive magnetomotive optical coherence microscopy (MM-OCM) and multiphoton microscopy (MPM).MPMis utilized for the visualization of multifunctional fluorescent and magnetic microbeads, while MM-OCM detects, with nanometer-scale sensitivity, periodic displacements of the microbeads induced by the modulation of an external magnetic field. Magnetomotive signals are measured from mouse macrophages, human breast primary ductal carcinoma cells, and human breast epithelial cells in culture, and validated with full-field phase-sensitive microscopy. This methodology demonstrates the capability for imaging controlled cell dynamics and has the potential for measuring cell biomechanical properties, which are important in assessing the health and pathological state of cells. PMID:25400496

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

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

    PubMed

    Tam, Johnny; Merino, David

    2015-11-01

    Stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) microscopy are two super-resolution optical microscopy approaches that have rapidly gained popularity in recent years. Both modalities offer super-resolution imaging capabilities with the potential for imaging in multiple colors, three-dimensions, and the possibility to image in live cells. In this review, we focus on the specific advantages and disadvantages of each technique in the context of each other. STORM has been reported to achieve higher spatial resolution when compared to STED, but a lengthy acquisition may be required. STED utilizes relatively higher laser intensities, but is able to generate a super-resolution image immediately after acquisition without the need for any additional data processing. Ultimately, the choice between STORM and STED will depend not only on the specific application, but also on the users' ability to understand and optimize the various parameters ranging from sample preparation to image acquisition, which determine the quality of the final image. Stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) are two super-resolution microscopy approaches that have rapidly gained popularity in recent years. STORM is based on the precise localization of a large number of individual molecules that together form a super-resolved image (bottom), whereas STED is based on the scanning of two super-imposed light sources which together allow for a super-resolved spot on the sample to be imaged (top). We discuss the specific advantages and disadvantages of each technique and explain the various parameters that affect image quality, which should be taken into consideration when planning experiments. PMID:26222552

  19. Optical characters and texture maps of skin and the aging mechanism by use of multiphoton microscopy and optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Wu, Shulian; Li, Hui; Zhang, Xiaoman; Huang, Yudian; Xu, Xiaohui

    2012-03-01

    Cutaneous aging is a complicated biological process affecting different constituents of skin, which can be divided into two types: the chronological aging and the photo-aging. The two cutaneous aging processes often co-exist accompanying with each other. The effects are often overlapped including changes in epithelium and dermis. The degeneration of collagen is a major factor in dermal alteration with aging. In this study, multiphoton microscopy (MPM) with its high resolution imaging and optical coherence tomography (OCT) with its depth resolved imaging were used to study the anti-aging dermatology in vivo. It was attempted to make the optical parameter and texture feature to evaluate the process of aging skin using mathematical image processing. The links among optical parameter, spectrum and texture feature in collagen with aging process were established to uncover mechanism of aging skin.

  20. Cross-validation of interferometric synthetic aperture microscopy and optical coherence tomography.

    PubMed

    Ralston, Tyler S; Adie, Steven G; Marks, Daniel L; Boppart, Stephen A; Carney, P Scott

    2010-05-15

    Computationally reconstructed interferometric synthetic aperture microscopy is coregistered with optical coherence tomography (OCT) focal plane data to provide quantitative cross validation with OCT. This is accomplished through a qualitative comparison of images and a quantitative analysis of the width of the point-spread function in simulation and experiment. The width of the ISAM point-spread function is seen to be independent of depth, in contrast to OCT. PMID:20479849

  1. Linear and Nonlinear Optical Spectroscopy at the Nanoscale with Photoinduced Force Microscopy.

    PubMed

    Jahng, Junghoon; Fishman, Dmitry A; Park, Sung; Nowak, Derek B; Morrison, Will A; Wickramasinghe, H Kumar; Potma, Eric O

    2015-10-20

    The enormous advances made in nanotechnology have also intensified the need for tools that can characterize newly synthesized nanoaterials with high sensitivity and with high spatial resolution. Many existing tools with nanoscopic resolution or better, including scanning electron microscopy (SEM), atomic force microscopy (AFM), and scanning tunneling microscopy (STM) methods, can generate highly detailed maps of nanoscopic structures. However, while these approaches provide great views of the morphological properties of nanomaterials, it has proven more challenging to derive chemical information from the corresponding images. To address this issue, attempts have been made to dress existing nanoscopy methods with spectroscopic sensitivity. A powerful approach in this direction is the combination of scan probe techniques with optical illumination, which aims to marry the nanoscopic resolution provided by a sharp tip with the chemical selectivity provided by optical spectroscopy. Examples of this approach include existing techniques such as scattering-type scanning near-field optical microscopy and tip-enhanced Raman spectroscopy. A new and emerging technique in this direction is photoinduced force microscopy (PiFM), which enables spectroscopic probing of materials with a spatial resolution well under 10 nm. In PiFM, the sample is optically excited and the response of the material is probed directly in the near-field by reading out the time-integrated force between the tip and the sample. Because the magnitude of the force is dependent on the photoinduced polarization in the sample, PiFM exhibits spectroscopic sensitivity. The photoinduced forces measured in PiFM are spatially confined on the nanometer scale, which translates into a very high spatial resolution even under ambient conditions. The PiFM approach is compatible with a wide range optical excitation frequencies, from the visible to the mid-infrared, enabling nanoscale imaging contrast based on either

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

    NASA Astrophysics Data System (ADS)

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

    2011-01-01

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

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

    PubMed

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

    2011-01-01

    We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish. PMID:21280920

  4. Optical tweezers and multiphoton microscopies integrated photonic tool for mechanical and biochemical cell processes studies

    NASA Astrophysics Data System (ADS)

    de Thomaz, A. A.; Faustino, W. M.; Fontes, A.; Fernandes, H. P.; Barjas-Castro, M. d. L.; Metze, K.; Giorgio, S.; Barbosa, L. C.; Cesar, C. L.

    2007-09-01

    The research in biomedical photonics is clearly evolving in the direction of the understanding of biological processes at the cell level. The spatial resolution to accomplish this task practically requires photonics tools. However, an integration of different photonic tools and a multimodal and functional approach will be necessary to access the mechanical and biochemical cell processes. This way we can observe mechanicaly triggered biochemical events or biochemicaly triggered mechanical events, or even observe simultaneously mechanical and biochemical events triggered by other means, e.g. electricaly. One great advantage of the photonic tools is its easiness for integration. Therefore, we developed such integrated tool by incorporating single and double Optical Tweezers with Confocal Single and Multiphoton Microscopies. This system can perform 2-photon excited fluorescence and Second Harmonic Generation microscopies together with optical manipulations. It also can acquire Fluorescence and SHG spectra of specific spots. Force, elasticity and viscosity measurements of stretched membranes can be followed by real time confocal microscopies. Also opticaly trapped living protozoas, such as leishmania amazonensis. Integration with CARS microscopy is under way. We will show several examples of the use of such integrated instrument and its potential to observe mechanical and biochemical processes at cell level.

  5. Motion capture and manipulation of a single synthetic molecular rotor by optical microscopy.

    PubMed

    Ikeda, Tomohiro; Tsukahara, Takahiro; Iino, Ryota; Takeuchi, Masayuki; Noji, Hiroyuki

    2014-09-15

    Single-molecule imaging and manipulation with optical microscopy have become essential methods for studying biomolecular machines; however, only few efforts have been directed towards synthetic molecular machines. Single-molecule optical microscopy was now applied to a synthetic molecular rotor, a double-decker porphyrin (DD). By attaching a magnetic bead (ca. 200 nm) to the DD, its rotational dynamics were captured with a time resolution of 0.5 ms. DD showed rotational diffusion with 90° steps, which is consistent with its four-fold structural symmetry. Kinetic analysis revealed the first-order kinetics of the 90° step with a rate constant of 2.8 s(-1). The barrier height of the rotational potential was estimated to be greater than 7.4 kJ mol(-1) at 298 K. The DD was also forcibly rotated with magnetic tweezers, and again, four stable pausing angles that are separated by 90° were observed. These results demonstrate the potency of single-molecule optical microscopy for the elucidation of elementary properties of synthetic molecular machines. PMID:24989127

  6. Measurements of adipose derived stem cell vitality with optical coherence phase microscopy

    NASA Astrophysics Data System (ADS)

    Bagnaninchi, P. O.; Holmes, C.; Drummond, N.; Daoud, J.; Tabrizian, M.

    2011-03-01

    Live cells display a constant vertical motility due partly to a constant rearrangement of focal contacts and to cell shape fluctuations. This cellular micromotion has been clearly demonstrated with electric cell impedance sensing (ECIS) on 2D micro-electrodes, and correlated to cell vitality. In this study we investigated if optical coherence phase microscopy (OCPM) was able to report phase fluctuations of adult stem cells in 2D and 3D that could be correlated to cell motility. An OCPM has been developed around a Thorlabs engine (λο=930nm FWHM: 90nm) and integrated in an inverted microscope with a custom scanning head. Human adipose derived stem cells (ADSCs, Invitrogen) were cultured in Mesenpro RS medium and seeded either on ECIS arrays, 2D cell culture dishes, or in 3D highly porous microplotted polymeric scaffolds. ADSC motility was measured by ECIS and a spectral analysis was performed to retrieve the power spectral density (PSD) of the fluctuations. Cells in standard media and fixed cells were investigated. The same conditions were then investigated for ADSCs in 2D and in 3D with optical coherence phase microscopy. Significant differences were found in phase fluctuations between the different conditions, which correlated well with ECIS experiments. These preliminary results indicated that optical coherence phase microscopy could assess cell vitality in 2D and potentially in 3D microstructures.

  7. Identification and evaluation on the phagocytic function of human neutrophils in diabetic patients by optical microscopy with cellular monolayer techniques and electron microscopy

    NASA Astrophysics Data System (ADS)

    Yu, Ye-Rong; Liang, Jing-Zhong

    1995-01-01

    A comparative study on phagocytosis of P. Aeruginosa by human neutrophils in diabetic patients and healthy volunteers was carried out by means of the monolayer of neutrophils in optical microscopy and ultrastructural observation in electronic microscopy. The results demonstrated that the level of phagocytosis in diabetics is lower than health people. The impairment in phagocytosis of neutrophils may be the important cause of severe and repeated infection in diabetic patients.

  8. The development of optical microscopy techniques for the advancement of single-particle studies

    NASA Astrophysics Data System (ADS)

    Marchuk, Kyle

    Single particle orientation and rotational tracking (SPORT) has recently become a powerful optical microscopy tool that can expose many molecular motions. Unfortunately, there is not yet a single microscopy technique that can decipher all particle motions in all environmental conditions, thus there are limitations to current technologies. Within, the two powerful microscopy tools of total internal reflection and interferometry are advanced to determine the position, orientation, and optical properties of metallic nanoparticles in a variety of environments. Total internal reflection is an optical phenomenon that has been applied to microscopy to produce either fluorescent or scattered light. The non-invasive far-field imaging technique is coupled with a near-field illumination scheme that allows for better axial resolution than confocal microscopy and epi-fluorescence microscopy. By controlling the incident illumination angle using total internal reflection fluorescence (TIRF) microscopy, a new type of imaging probe called "non-blinking" quantum dots (NBQDs) were super-localized in the axial direction to sub-10-nm precision. These particles were also used to study the rotational motion of microtubules being propelled by the motor protein kinesin across the substrate surface. The same instrument was modified to function under total internal reflection scattering (TIRS) microscopy to study metallic anisotropic nanoparticles and their dynamic interactions with synthetic lipid bilayers. Utilizing two illumination lasers with opposite polarization directions at wavelengths corresponding to the short and long axis surface plasmon resonance (SPR) of the nanoparticles, both the in-plane and out-of-plane movements of many particles could be tracked simultaneously. When combined with Gaussian point spread function (PSF) fitting for particle super-localization, the binding status and rotational movement could be resolved without degeneracy. TIRS microscopy was also used to

  9. The development of optical microscopy techniques for the advancement of single-particle studies

    SciTech Connect

    Marchuk, Kyle

    2013-05-15

    Single particle orientation and rotational tracking (SPORT) has recently become a powerful optical microscopy tool that can expose many molecular motions. Unfortunately, there is not yet a single microscopy technique that can decipher all particle motions in all environmental conditions, thus there are limitations to current technologies. Within, the two powerful microscopy tools of total internal reflection and interferometry are advanced to determine the position, orientation, and optical properties of metallic nanoparticles in a variety of environments. Total internal reflection is an optical phenomenon that has been applied to microscopy to produce either fluorescent or scattered light. The non-invasive far-field imaging technique is coupled with a near-field illumination scheme that allows for better axial resolution than confocal microscopy and epi-fluorescence microscopy. By controlling the incident illumination angle using total internal reflection fluorescence (TIRF) microscopy, a new type of imaging probe called “non-blinking” quantum dots (NBQDs) were super-localized in the axial direction to sub-10-nm precision. These particles were also used to study the rotational motion of microtubules being propelled by the motor protein kinesin across the substrate surface. The same instrument was modified to function under total internal reflection scattering (TIRS) microscopy to study metallic anisotropic nanoparticles and their dynamic interactions with synthetic lipid bilayers. Utilizing two illumination lasers with opposite polarization directions at wavelengths corresponding to the short and long axis surface plasmon resonance (SPR) of the nanoparticles, both the in-plane and out-of-plane movements of many particles could be tracked simultaneously. When combined with Gaussian point spread function (PSF) fitting for particle super-localization, the binding status and rotational movement could be resolved without degeneracy. TIRS microscopy was also used to

  10. Dynamic multicomponent engineered tissue reorganization and matrix deposition measured with an integrated nonlinear optical microscopy–optical coherence microscopy system

    PubMed Central

    Bai, Yuqiang; Lee, Po-Feng; Gibbs, Holly C.; Bayless, Kayla J.; Yeh, Alvin T.

    2014-01-01

    Abstract. Multicomponent tissue models are viable tools to better understand cell responses in complex environments, but present challenges when investigated with live cell microscopy noninvasively. In this study, integrated nonlinear optical microscopy-optical coherence microscopy (NLOM-OCM) was used to characterize cell interactions within three-dimensional (3-D), multicomponent extracellular matrices. In fibrin-collagen mixtures, 3T3 fibroblasts were observed to recruit both fibrin and collagen fibers while remodeling matrices. Also, NLOM-OCM was used to observe collagen deposition by neonatal human dermal fibroblasts within originally fibrin matrices over an extended time. It was observed that preferentially aligned collagen deposition could be achieved with aligned fibroblasts but that cell alignment could be achieved without aligning the extant extracellular matrix. In summary, this multimodel imaging system has potential for both real-time and longitudinal imaging of living 3-D cultures, which is particularly important for evaluating cell microenvironments in composite scaffolds or serial characterization of engineered tissue constructs during culture. PMID:24647972

  11. Scattering-type scanning near-field optical microscopy with reconstruction of vertical interaction.

    PubMed

    Wang, Le; Xu, Xiaoji G

    2015-01-01

    Scattering-type scanning near-field optical microscopy provides access to super-resolution spectroscopic imaging of the surfaces of a variety of materials and nanostructures. In addition to chemical identification, it enables observations of nano-optical phenomena, such as mid-infrared plasmons in graphene and phonon polaritons in boron nitride. Despite the high lateral spatial resolution, scattering-type near-field optical microscopy is not able to provide characteristics of near-field responses in the vertical dimension, normal to the sample surface. Here, we present an accurate and fast reconstruction method to obtain vertical characteristics of near-field interactions. For its first application, we investigated the bound electromagnetic field component of surface phonon polaritons on the surface of boron nitride nanotubes and found that it decays within 20 nm with a considerable phase change in the near-field signal. The method is expected to provide characterization of the vertical field distribution of a wide range of nano-optical materials and structures. PMID:26592949

  12. Scattering-type scanning near-field optical microscopy with reconstruction of vertical interaction

    NASA Astrophysics Data System (ADS)

    Wang, Le; Xu, Xiaoji G.

    2015-11-01

    Scattering-type scanning near-field optical microscopy provides access to super-resolution spectroscopic imaging of the surfaces of a variety of materials and nanostructures. In addition to chemical identification, it enables observations of nano-optical phenomena, such as mid-infrared plasmons in graphene and phonon polaritons in boron nitride. Despite the high lateral spatial resolution, scattering-type near-field optical microscopy is not able to provide characteristics of near-field responses in the vertical dimension, normal to the sample surface. Here, we present an accurate and fast reconstruction method to obtain vertical characteristics of near-field interactions. For its first application, we investigated the bound electromagnetic field component of surface phonon polaritons on the surface of boron nitride nanotubes and found that it decays within 20 nm with a considerable phase change in the near-field signal. The method is expected to provide characterization of the vertical field distribution of a wide range of nano-optical materials and structures.

  13. Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging

    PubMed Central

    Abrahamsson, Sara; Ilic, Rob; Wisniewski, Jan; Mehl, Brian; Yu, Liya; Chen, Lei; Davanco, Marcelo; Oudjedi, Laura; Fiche, Jean-Bernard; Hajj, Bassam; Jin, Xin; Pulupa, Joan; Cho, Christine; Mir, Mustafa; El Beheiry, Mohamed; Darzacq, Xavier; Nollmann, Marcelo; Dahan, Maxime; Wu, Carl; Lionnet, Timothée; Liddle, J. Alexander; Bargmann, Cornelia I.

    2016-01-01

    Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a “precise color” MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is ≈90% and we have obtained efficiencies of > 80% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living C. elegans. PMID:27231594

  14. Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging.

    PubMed

    Abrahamsson, Sara; Ilic, Rob; Wisniewski, Jan; Mehl, Brian; Yu, Liya; Chen, Lei; Davanco, Marcelo; Oudjedi, Laura; Fiche, Jean-Bernard; Hajj, Bassam; Jin, Xin; Pulupa, Joan; Cho, Christine; Mir, Mustafa; El Beheiry, Mohamed; Darzacq, Xavier; Nollmann, Marcelo; Dahan, Maxime; Wu, Carl; Lionnet, Timothée; Liddle, J Alexander; Bargmann, Cornelia I

    2016-03-01

    Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a "precise color" MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is ≈90% and we have obtained efficiencies of > 80% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living C. elegans. PMID:27231594

  15. Scattering-type scanning near-field optical microscopy with reconstruction of vertical interaction

    PubMed Central

    Wang, Le; Xu, Xiaoji G.

    2015-01-01

    Scattering-type scanning near-field optical microscopy provides access to super-resolution spectroscopic imaging of the surfaces of a variety of materials and nanostructures. In addition to chemical identification, it enables observations of nano-optical phenomena, such as mid-infrared plasmons in graphene and phonon polaritons in boron nitride. Despite the high lateral spatial resolution, scattering-type near-field optical microscopy is not able to provide characteristics of near-field responses in the vertical dimension, normal to the sample surface. Here, we present an accurate and fast reconstruction method to obtain vertical characteristics of near-field interactions. For its first application, we investigated the bound electromagnetic field component of surface phonon polaritons on the surface of boron nitride nanotubes and found that it decays within 20 nm with a considerable phase change in the near-field signal. The method is expected to provide characterization of the vertical field distribution of a wide range of nano-optical materials and structures. PMID:26592949

  16. Enhancement of optical coherence microscopy in turbid media by an optical parametric amplifier.

    PubMed

    Zhao, Youbo; Tu, Haohua; Liu, Yuan; Bower, Andrew J; Boppart, Stephen A

    2015-06-01

    We report the enhancement in imaging performance of a spectral-domain optical coherence microscope (OCM) in turbid media by incorporating an optical parametric amplifier (OPA). The OPA provides a high level of optical gain to the sample arm, thereby improving the signal-to-noise ratio of the OCM by a factor of up to 15 dB. A unique nonlinear confocal gate is automatically formed in the OPA, which enables selective amplification of singly scattered (ballistic) photons against the multiply-scattered light background. Simultaneous enhancement in both imaging depth and spatial resolution in imaging microstructures in highly light-scattering media are demonstrated with the combined OPA-OCM setup. Typical OCM inteferograms (left) and images (right) without and with OPA. PMID:25196251

  17. Photonic Torque Microscopy of the Nonconservative Force Field for Optically Trapped Silicon Nanowires.

    PubMed

    Irrera, Alessia; Magazzù, Alessandro; Artoni, Pietro; Simpson, Stephen H; Hanna, Simon; Jones, Philip H; Priolo, Francesco; Gucciardi, Pietro Giuseppe; Maragò, Onofrio M

    2016-07-13

    We measure, by photonic torque microscopy, the nonconservative rotational motion arising from the transverse components of the radiation pressure on optically trapped, ultrathin silicon nanowires. Unlike spherical particles, we find that nonconservative effects have a significant influence on the nanowire dynamics in the trap. We show that the extreme shape of the trapped nanowires yields a transverse component of the radiation pressure that results in an orbital rotation of the nanowire about the trap axis. We study the resulting motion as a function of optical power and nanowire length, discussing its size-scaling behavior. These shape-dependent nonconservative effects have implications for optical force calibration and optomechanics with levitated nonspherical particles. PMID:27280642

  18. Label-free optical imaging of membrane patches for atomic force microscopy

    PubMed Central

    Churnside, Allison B.; King, Gavin M.; Perkins, Thomas T.

    2010-01-01

    In atomic force microscopy (AFM), finding sparsely distributed regions of interest can be difficult and time-consuming. Typically, the tip is scanned until the desired object is located. This process can mechanically or chemically degrade the tip, as well as damage fragile biological samples. Protein assemblies can be detected using the back-scattered light from a focused laser beam. We previously used back-scattered light from a pair of laser foci to stabilize an AFM. In the present work, we integrate these techniques to optically image patches of purple membranes prior to AFM investigation. These rapidly acquired optical images were aligned to the subsequent AFM images to ~40 nm, since the tip position was aligned to the optical axis of the imaging laser. Thus, this label-free imaging efficiently locates sparsely distributed protein assemblies for subsequent AFM study while simultaneously minimizing degradation of the tip and the sample. PMID:21164738

  19. Optical-Resolution Photoacoustic Microscopy: Auscultation of Biological Systems at the Cellular Level

    PubMed Central

    Hu, Song; Wang, Lihong V.

    2013-01-01

    Photoacoustic microscopy (PAM) offers unprecedented sensitivity to optical absorption and opens a new window to study biological systems at multiple length- and timescales. In particular, optical-resolution PAM (OR-PAM) has pushed the technical envelope to submicron length scales and millisecond timescales. Here, we review the state of the art of OR-PAM in biophysical research. With properly chosen optical wavelengths, OR-PAM can spectrally differentiate a variety of endogenous and exogenous chromophores, unveiling the anatomical, functional, metabolic, and molecular information of biological systems. Newly uncovered contrast mechanisms of linear dichroism and Förster resonance energy transfer further distinguish OR-PAM. Integrating multiple contrasts and advanced scanning mechanisms has capacitated OR-PAM to comprehensively interrogate biological systems at the cellular level in real time. Two future directions are discussed, where OR-PAM holds the potential to translate basic biophysical research into clinical healthcare. PMID:23972836

  20. All-optical photoacoustic microscopy based on plasmonic detection of broadband ultrasound

    NASA Astrophysics Data System (ADS)

    Wang, Tianxiong; Cao, Rui; Ning, Bo; Dixon, Adam J.; Hossack, John A.; Klibanov, Alexander L.; Zhou, Qifa; Wang, Anbo; Hu, Song

    2015-10-01

    We report on an implementation of all-optical photoacoustic microscopy (PAM), which capitalizes on the effect of surface plasmon resonance (SPR) for optical detection of ultrasound. The SPR sensor in our all-optical PAM shows, experimentally, a linear response to the acoustic pressure from 5.2 kPa to 2.1 MPa, an ultra-flat frequency response (±0.7 dB) from 680 kHz to 126 MHz, and a noise-equivalent pressure sensitivity of 3.3 kPa. With the broadband ultrasonic detection, our SPR-PAM has achieved high spatial resolution with relatively low anisotropy (i.e., 2.0 μm laterally and 8.4 μm axially). Three-dimensional high-resolution imaging of a single melanoma cell is demonstrated.

  1. Field of view advantage of conjugate adaptive optics in microscopy applications

    PubMed Central

    Mertz, Jerome; Paudel, Hari; Bifano, Thomas G.

    2015-01-01

    The imaging performance of an optical microscope can be degraded by sample-induced aberrations. A general strategy to undo the effect of these aberrations is to apply wavefront correction with a deformable mirror (DM). In most cases the DM is placed conjugate to the microscope pupil, called pupil adaptive optics (AO). When the aberrations are spatially variant an alternative configuration involves placing the DM conjugate to the main source of aberrations, called conjugate AO. We provide a theoretical and experimental comparison of both configurations for the simplified case where spatially variant aberrations are produced by a well defined phase screen. We pay particular attention to the resulting correction field of view (FOV). Conjugate AO is found to provide a significant FOV advantage. While this result is well known in the astronomy community, our goal here is to recast it specifically for the optical microscopy community. PMID:25967343

  2. High numerical aperture injection-molded miniature objective for fiber-optic confocal reflectance microscopy

    NASA Astrophysics Data System (ADS)

    Chidley, Matthew Douglas

    This dissertation presents the design of a miniature injection-molded objective lens for a fiber-optic confocal reflectance microscope. This is part of an effort to demonstrate the ability to fabricate low cost, high performance biomedical optics for high resolution in vivo imaging. Disposable endoscopic microscope objectives could help in vivo confocal microscopy technology mature to enable large-scale clinical screening and detection of early cancers and pre-cancerous lesions. This five lens plastic objective has been tested as a stand-alone optical system and has been coupled to a confocal microscope for in vivo imaging of cells and tissue. Changing the spacing and rotation of the individual optical elements can compensate for fabrication inaccuracies and improve performance. An optical-bench testing system was constructed to allow interactive alignment during testing. The modulation transfer function (MTF) of the miniature objective lens is determined using the slanted-edge method. A custom MATLAB program, edgeMTF, was written to collect, analyze, and record test data. An estimated Strehl ratio of 0.64 and an MTF value of 0.70, at the fiber-optic bundle Nyquist frequency, have been obtained. The main performance limitations of the miniature objective are mechanical alignment and flow-induced birefringence. Annealing and experimental injection molding runs were conducted in effort to reduce birefringence.

  3. Ex vivo blood vessel imaging using ultrasound-modulated optical microscopy

    PubMed Central

    Kothapalli, Sri-Rajasekhar; Wang, Lihong V.

    2009-01-01

    Recently we developed ultrasound-modulated optical microscopy (UOM) based on a long-cavity confocal Fabry-Perot interferometer (CFPI) [J. Biomed. Opt. 13(5), 0504046, (2008)]. This interferometer is used for real time detection of multiply scattered light modulated by high frequency (30 MHz to 75 MHz) ultrasound pulses propagating in an optically strongly scattering medium. In this article, we use this microscope to study the dependence of ultrasound-modulated optical signals on the optical absorption and scattering properties of objects embedded about 3 mm deep in tissue mimicking phantoms. These results demonstrate that UOM has the potential to map both optical absorption and scattering contrast. Most importantly, for the first time in the field of ultrasound-modulated optical imaging, we imaged blood vasculature in highly scattering tissue samples from a mouse and a rat. Therefore UOM could be a promising tool to study the morphology of blood vasculature and blood-associated functional parameters, such as oxygen saturation. PMID:19256703

  4. Astrocytes in the optic nerve head express putative mechanosensitive channels

    PubMed Central

    Choi, Hee Joo; Sun, Daniel

    2015-01-01

    Purpose To establish whether optic nerve head astrocytes express candidate molecules to sense tissue stretch. Methods We used conventional PCR, quantitative PCR, and single-cell reverse transcription PCR (RT–PCR) to assess the expression of various members of the transient receptor potential (TRP) channel family and of the recently characterized mechanosensitive channels Piezo1 and 2 in optic nerve head tissue and in single, isolated astrocytes. Results Most TRP subfamilies (TRPC, TRPM, TRPV, TRPA, and TRPP) and Piezo1 and 2 were expressed in the optic nerve head of the mouse. Quantitative real-time PCR analysis showed that TRPC1, TRPM7, TRPV2, TRPP2, and Piezo1 are the dominant isoforms in each subfamily. Single-cell RT–PCR revealed that many TRP isoforms, TRPC1–2, TRPC6, TRPV2, TRPV4, TRPM2, TRPM4, TRPM6–7, TRPP1–2, and Piezo1–2, are expressed in astrocytes of the optic nerve head, and that most astrocytes express TRPC1 and TRPP1–2. Comparisons of the TRPP and Piezo expression levels between different tissue regions showed that Piezo2 expression was higher in the optic nerve head and the optic nerve proper than in the brain and the corpus callosum. TRPP2 also showed higher expression in the optic nerve head. Conclusions Astrocytes in the optic nerve head express multiple putative mechanosensitive channels, in particular the recently identified channels Piezo1 and 2. The expression of putative mechanosensitive channels in these cells may contribute to their responsiveness to traumatic or glaucomatous injury. PMID:26236150

  5. Fast and robust optical flow for time-lapse microscopy using super-voxels

    PubMed Central

    Amat, Fernando; Myers, Eugene W.; Keller, Philipp J.

    2013-01-01

    Motivation: Optical flow is a key method used for quantitative motion estimation of biological structures in light microscopy. It has also been used as a key module in segmentation and tracking systems and is considered a mature technology in the field of computer vision. However, most of the research focused on 2D natural images, which are small in size and rich in edges and texture information. In contrast, 3D time-lapse recordings of biological specimens comprise up to several terabytes of image data and often exhibit complex object dynamics as well as blurring due to the point-spread-function of the microscope. Thus, new approaches to optical flow are required to improve performance for such data. Results: We solve optical flow in large 3D time-lapse microscopy datasets by defining a Markov random field (MRF) over super-voxels in the foreground and applying motion smoothness constraints between super-voxels instead of voxel-wise. This model is tailored to the specific characteristics of light microscopy datasets: super-voxels help registration in textureless areas, the MRF over super-voxels efficiently propagates motion information between neighboring cells and the background subtraction and super-voxels reduce the dimensionality of the problem by an order of magnitude. We validate our approach on large 3D time-lapse datasets of Drosophila and zebrafish development by analyzing cell motion patterns. We show that our approach is, on average, 10 × faster than commonly used optical flow implementations in the Insight Tool-Kit (ITK) and reduces the average flow end point error by 50% in regions with complex dynamic processes, such as cell divisions. Availability: Source code freely available in the Software section at http://janelia.org/lab/keller-lab. Contact: amatf@janelia.hhmi.org or kellerp@janelia.hhmi.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23242263

  6. Reciprocity theory of apertureless scanning near-field optical microscopy with point-dipole probes.

    PubMed

    Esslinger, Moritz; Vogelgesang, Ralf

    2012-09-25

    Near-field microscopy offers the opportunity to reveal optical contrast at deep subwavelength scales. In scanning near-field optical microscopy (SNOM), the diffraction limit is overcome by a nanoscopic probe in close proximity to the sample. The interaction of the probe with the sample fields necessarily perturbs the bare sample response, and a critical issue is the interpretation of recorded signals. For a few specific SNOM configurations, individual descriptions have been modeled, but a general and intuitive framework is still lacking. Here, we give an exact formulation of the measurable signals in SNOM which is easily applicable to experimental configurations. Our results are in close analogy with the description Tersoff and Hamann have derived for the tunneling currents in scanning tunneling microscopy. For point-like scattering probe tips, such as used in apertureless SNOM, the theory simplifies dramatically to a single scalar relation. We find that the measured signal is directly proportional to the field of the coupled tip-sample system at the position of the tip. For weakly interacting probes, the model thus verifies the empirical findings that the recorded signal is proportional to the unperturbed field of the bare sample. In the more general case, it provides guidance to an intuitive and faithful interpretation of recorded images, facilitating the characterization of tip-related distortions and the evaluation of novel SNOM configurations, both for aperture-based and apertureless SNOM. PMID:22897563

  7. Deep-sea spherules from Pacific clay - Mass distribution and influx rate. [extraterrestrial origins from optical and electron microscopy

    NASA Technical Reports Server (NTRS)

    Murrell, M. T.; Davis, P. A., Jr.; Nishiizumi, K.; Millard, H. T., Jr.

    1980-01-01

    From 411 kg of Pacific clay, 22 mg of stony spherules and 50 mg of iron spherules larger than 150 microns were concentrated. The extraterrestrial origin of these particles was evaluated with the aid of optical and electron microscopy and atomic absorption elemental analysis. An expression for the integral number of stony particles from this sediment in the mass range 20-300 micrograms was derived. The world-wide influx rate of stony particles in the mass range which survive atmospheric heating and ocean sediment storage is calculated to be 90 tons/yr. The relative contributions of ablation debris vs fused interplanetary dust to the influx of stony spherules is discussed, but no conclusions could be made.

  8. Finite-difference time-domain-based optical microscopy simulation of dispersive media facilitates the development of optical imaging techniques

    NASA Astrophysics Data System (ADS)

    Zhang, Di; Capoglu, Ilker; Li, Yue; Cherkezyan, Lusik; Chandler, John; Spicer, Graham; Subramanian, Hariharan; Taflove, Allen; Backman, Vadim

    2016-06-01

    Combining finite-difference time-domain (FDTD) methods and modeling of optical microscopy modalities, we previously developed an open-source software package called Angora, which is essentially a "microscope in a computer." However, the samples being simulated were limited to nondispersive media. Since media dispersions are common in biological samples (such as cells with staining and metallic biomarkers), we have further developed a module in Angora to simulate samples having complicated dispersion properties, thereby allowing the synthesis of microscope images of most biological samples. We first describe a method to integrate media dispersion into FDTD, and we validate the corresponding Angora dispersion module by applying Mie theory, as well as by experimentally imaging gold microspheres. Then, we demonstrate how Angora can facilitate the development of optical imaging techniques with a case study.

  9. Miniature injection-molded optics for fiber-optic, in vivo confocal microscopy

    NASA Astrophysics Data System (ADS)

    Chidley, Matthew D.; Liang, Chen; Descour, Michael R.; Sung, Kung-Bin; Richards-Kortum, Rebecca R.; Gillenwater, Ann

    2002-12-01

    In collaboration with the Department of Biomedical Engineering at the University of Texas at Austin and the UT MD Anderson Cancer Center, a laser scanning fiber confocal reflectance microscope (FCRM) system has been designed and tested for in vivo detection of cervical and oral pre-cancers. This system along with specially developed diagnosis algorithms and techniques can achieve an unprecedented specificity and sensitivity for the diagnosis of pre-cancers in epithelial tissue. The FCRM imaging system consists of an NdYAG laser (1064 nm), scanning mirrors/optics, precision pinhole, detector, and an endoscopic probe (the objective). The objective is connected to the rest of the imaging system via a fiber bundle. The fiber bundle allows the rest of the system to be remotely positioned in a convenient location. Only the objective comes into contact with the patient. It is our intent that inexpensive mass-produced disposable endoscopic probes would be produced for large clinical trials. This paper touches on the general design process of developing a miniature, high numerical aperture, injection-molded (IM) objective. These IM optical designs are evaluated and modified based on manufacturing and application constraints. Based on these driving criteria, one specific optical design was chosen and a detailed tolerance analysis was conducted. The tolerance analysis was custom built to create a realistic statistical analysis for integrated IM lens elements that can be stacked one on top of another using micro-spheres resting in tiny circular grooves. These configurations allow each lens element to be rotated and possibly help compensate for predicted manufacturing errors. This research was supported by a grant from the National Institutes of Health (RO1 CA82880). Special thanks go to Applied Image Group/Optics for the numerous fabrication meetings concerning the miniature IM objective.

  10. Gold nanoparticle tips for optical field confinement in infrared scattering near-field optical microscopy.

    PubMed

    Wenzel, Marc Tobias; Härtling, Thomas; Olk, Phillip; Kehr, Susanne C; Grafström, Stefan; Winnerl, Stephan; Helm, Manfred; Eng, Lukas M

    2008-08-01

    We report on the implementation of metal nanoparticles as probes for scattering and apertureless near-field optical investigations in the mid-infrared (mid-IR) spectral regime. At these wavelengths, an efficient electric-field confinement is necessary and achieved here through a gold metal nanoparticle of 80 nm in diameter (Au80-MNP) acting as the optical antenna. The Au80-MNP is attached to a standard AFM cantilever used as the spatial manipulator. When approached to a sample surface while being illuminated with an infrared beam, the Au80-MNP produces a considerably improved spatial confinement of the electric field compared to an ordinary scattering AFM tip. We demonstrate here the confinement normal to the sample surface by making use of a sample-induced phonon polariton resonance in a ferroelectric lithium niobate sample. Our experimental findings are in very good agreement with the quasistatic dipole model and show improved optical resolution via well-selected antenna particles. PMID:18679508

  11. Topographic, electrochemical, and optical images captured using standing approach mode scanning electrochemical/optical microscopy.

    PubMed

    Takahashi, Yasufumi; Hirano, Yu; Yasukawa, Tomoyuki; Shiku, Hitoshi; Yamada, Hiroshi; Matsue, Tomokazu

    2006-12-01

    We developed a high-resolution scanning electrochemical microscope (SECM) for the characterization of various biological materials. Electrode probes were fabricated by Ti/Pt sputtering followed by parylene C-vapor deposition polymerization on the pulled optical fiber or glass capillary. The effective electrode radius estimated from the cyclic voltammogram of ferrocyanide was found to be 35 nm. The optical aperture size was less than 170 nm, which was confirmed from the cross section of the near-field scanning optical microscope (NSOM) image of the quantum dot (QD) particles with diameters in the range of 10-15 nm. The feedback mechanism controlling the probe-sample distance was improved by vertically moving the probe by 0.1-3 microm to reduce the damage to the samples. This feedback mode, defined as "standing approach (STA) mode" (Yamada, H.; Fukumoto, H.; Yokoyama, T.; Koike, T. Anal. Chem. 2005, 77, 1785-1790), has allowed the simultaneous electrochemical and topographic imaging of the axons and cell body of a single PC12 cell under physiological conditions for the first time. STA-mode feedback imaging functions better than tip-sample regulation by the conventionally available AFM. For example, polystyrene beads (diameter approximately 6 microm) was imaged using the STA-mode SECM, whereas imaging was not possible using a conventional AFM instrument. PMID:17128996

  12. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

    PubMed Central

    Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-01-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43− symmetric stretch vibrations at 959 cm−1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis. PMID:27225821

  13. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

    NASA Astrophysics Data System (ADS)

    Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-05-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43‑ symmetric stretch vibrations at 959 cm‑1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis.

  14. Characterization of atherosclerotic arterial tissue using multimodal non-linear optical microscopy

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

    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. Non-linear microscopy techniques have the potential to bridge this gap by providing morpho-functional information 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. The presented method has the potential to find a stable place in clinical setting as well as to be applied in vivo in the near future.

  15. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells.

    PubMed

    Hofemeier, Arne D; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F W; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-01-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO4(3-) symmetric stretch vibrations at 959 cm(-1) assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis. PMID:27225821

  16. Tri-modal microscopy with multiphoton and optical coherence microscopy/tomography for multi-scale and multi-contrast imaging

    PubMed Central

    Chong, Shau Poh; Lai, Tom; Zhou, Yifeng; Tang, Shuo

    2013-01-01

    Multi-scale multimodal microscopy is a very useful technique by providing multiple imaging contrasts with adjustable field of views and spatial resolutions. Here, we present a tri-modal microscope combining multiphoton microscopy (MPM), optical coherence microscopy (OCM) and optical coherence tomography (OCT) for subsurface visualization of biological tissues. The advantages of the tri-modal system are demonstrated on various biological samples. It enables the visualization of multiple intrinsic contrasts including scattering, two-photon excitation fluorescence (TPEF), and second harmonic generation (SHG). It also enables a rapid scanning over a large tissue area and a high resolution zoom-in for cellular-level structures on regions of interest. The tri-modal microscope can be important for label-free imaging to obtain a sufficient set of parameters for reliable sample analysis. PMID:24049679

  17. Light-sheet photoacoustic microscopy (LIS-PAM) with optical ultrasound detection

    NASA Astrophysics Data System (ADS)

    Nuster, Robert; Slezak, Paul; Paltauf, Guenther

    2016-03-01

    Photoacoustic (or optoacoustic) microscopy has great potential as a diagnostic tool in biomedical research. For in vivo imaging, an important requirement is to keep the measurement time as short as possible. In light-sheet photoacoustic microscopy (LIS-PAM) a cylindrical lens illuminates a thin section perpendicular to the sample surface with a short laser pulse and a projection of the excited acoustic wave pattern leaving the sample is recorded with a camera. From the recorded data, a B-scan photoacoustic image is obtained by applying a two-dimensional reconstruction algorithm, without requiring any mechanical scanning. Hence, LIS-PAM is capable of real-time B-scan imaging with acoustical resolution within the individual B-scans and optical out of plane resolution up to a depth limited by optical diffusion. A 3D image is composed of reconstructed B-scan images recorded while scanning the excitation line along the sample surface. Using a camera with 200 Hz frame rate a C-scan image (5x5 mm2 field of view) can be recorded in less than 5 seconds (without averaging). The achievable sensitivity and resolution of the optical phase contrast detection system were estimated theoretically with 0.34 kPa mm without averaging and 30 μm, respectively. A first experiment on a phantom that mimics tissue properties shows the applicability of this technique for in-vivo imaging.

  18. Optical microscopy as a comparative analytical technique for single-particle dissolution studies.

    PubMed

    Svanbäck, Sami; Ehlers, Henrik; Yliruusi, Jouko

    2014-07-20

    Novel, simple and cost effective methods are needed to replace advanced chemical analytical techniques, in small-scale dissolution studies. Optical microscopy of individual particles could provide such a method. The aim of the present work was to investigate and verify the applicability of optical microscopy as an analytical technique for drug dissolution studies. The evaluation was performed by comparing image and chemical analysis data of individual dissolving particles. It was shown that the data obtained by image analysis and UV-spectrophotometry produced practically identical dissolution curves, with average similarity and difference factors above 82 and below 4, respectively. The relative standard deviation for image analysis data, of the studied particle size range, varied between 1.9% and 3.8%. Consequently, it is proposed that image analysis can be used, on its own, as a viable analytical technique in single-particle dissolution studies. The possibility for significant reductions in sample preparation, operational cost, time and substance consumption gives optical detection a clear advantage over chemical analytical methods. Thus, image analysis could be an ideal and universal analytical technique for rapid small-scale dissolution studies. PMID:24751345

  19. Spectral interferometric microscopy reveals absorption by individual optical nanoantennas from extinction phase.

    PubMed

    Gennaro, Sylvain D; Sonnefraud, Yannick; Verellen, Niels; Van Dorpe, Pol; Moshchalkov, Victor V; Maier, Stefan A; Oulton, Rupert F

    2014-01-01

    Optical antennas transform light from freely propagating waves into highly localized excitations that interact strongly with matter. Unlike their radio frequency counterparts, optical antennas are nanoscopic and high frequency, making amplitude and phase measurements challenging and leaving some information hidden. Here we report a novel spectral interferometric microscopy technique to expose the amplitude and phase response of individual optical antennas across an octave of the visible to near-infrared spectrum. Although it is a far-field technique, we show that knowledge of the extinction phase allows quantitative estimation of nanoantenna absorption, which is a near-field quantity. To verify our method we characterize gold ring-disk dimers exhibiting Fano interference. Our results reveal that Fano interference only cancels a bright mode's scattering, leaving residual extinction dominated by absorption. Spectral interference microscopy has the potential for real-time and single-shot phase and amplitude investigations of isolated quantum and classical antennas with applications across the physical and life sciences. PMID:24781663

  20. Monitoring Volumetric Changes in Silicon Thin-Film Anodes through In Situ Optical Diffraction Microscopy.

    PubMed

    Duay, Jonathon; Schroder, Kjell W; Murugesan, Sankaran; Stevenson, Keith J

    2016-07-13

    A high-resolution in situ spectroelectrochemical optical diffraction experiment has been developed to understand the volume expansion/contraction process of amorphous silicon (a-Si) thin-film anodes. Electrodes consisting of 1D transmissive gratings of silicon have been produced through photolithographic methods. After glovebox assembly in a home-built Teflon cell, monitoring of the diffraction efficiency of these gratings during the lithiation/delithiation process is performed using an optical microscope equipped with a Bertrand lens. When the diffraction efficiency along with optical constants obtained from in situ spectroscopic ellipsometry is utilized, volume changes of the active materials can be deduced. Unlike transmission electron microscopy and atomic force microscopy characterization methods of observing silicon's volume expansion, this experiment allows for real-time monitoring of the volume change at charge/discharge cycles greater than just the first few along with an experimental environment that directly mimics that of a real battery. This technique shows promising results that provide needed insight into understanding the lithium alloying reaction and subsequent induced capacity fade during the cycling of alloying anodes in lithium-ion batteries. PMID:27311132

  1. Optical recording of action potentials with second-harmonic generation microscopy.

    PubMed

    Dombeck, Daniel A; Blanchard-Desce, Mireille; Webb, Watt W

    2004-01-28

    Nonlinear microscopy has proven to be essential for neuroscience investigations of thick tissue preparations. However, the optical recording of fast (approximately 1 msec) cellular electrical activity has never until now been successfully combined with this imaging modality. Through the use of second-harmonic generation microscopy of primary Aplysia neurons in culture labeled with 4-[4-(dihexylamino)phenyl][ethynyl]-1-(4-sulfobutyl)pyridinium (inner salt), we optically recorded action potentials with 0.833 msec temporal and 0.6 microm spatial resolution on soma and neurite membranes. Second-harmonic generation response as a function of change in membrane potential was found to be linear with a signal change of approximately 6%/100 mV. The signal-to-noise ratio was approximately 1 for single-trace action potential recordings but was readily increased to approximately 6-7 with temporal averaging of approximately 50 scans. Photodamage was determined to be negligible by observing action potential characteristics, cellular resting potential, and gross cellular morphology during and after laser illumination. High-resolution (micrometer scale) optical recording of membrane potential activity by previous techniques has been limited to imaging depths an order of magnitude less than nonlinear methods. Because second-harmonic generation is capable of imaging up to approximately 400 microm deep into intact tissue with submicron resolution and little out-of-focus photodamage or bleaching, its ability to record fast electrical activity should prove valuable to future electrophysiology studies. PMID:14749445

  2. Combined versatile high-resolution optical tweezers and single-molecule fluorescence microscopy

    PubMed Central

    Sirinakis, George; Ren, Yuxuan; Gao, Ying; Xi, Zhiqun; Zhang, Yongli

    2012-01-01

    Optical trapping and single-molecule fluorescence are two major single-molecule approaches. Their combination has begun to show greater capability to study more complex systems than either method alone, but met many fundamental and technical challenges. We built an instrument that combines base-pair resolution dual-trap optical tweezers with single-molecule fluorescence microscopy. The instrument has complementary design and functionalities compared with similar microscopes previously described. The optical tweezers can be operated in constant force mode for easy data interpretation or in variable force mode for maximum spatiotemporal resolution. The single-molecule fluorescence detection can be implemented in either wide-field or confocal imaging configuration. To demonstrate the capabilities of the new instrument, we imaged a single stretched λ DNA molecule and investigated the dynamics of a DNA hairpin molecule in the presence of fluorophore-labeled complementary oligonucleotide. We simultaneously observed changes in the fluorescence signal and pauses in fast extension hopping of the hairpin due to association and dissociation of individual oligonucleotides. The combined versatile microscopy allows for greater flexibility to study molecular machines or assemblies at a single-molecule level. PMID:23020384

  3. Multimodal and non-linear optical microscopy applications in reproductive biology.

    PubMed

    Adur, J; Barbosa, G O; Pelegati, V B; Baratti, M O; Cesar, C L; Casco, V H; Carvalho, H F

    2016-07-01

    A plethora of optical techniques is currently available to obtain non-destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four-dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed. Microsc. Res. Tech. 79:567-582, 2016. © 2016 Wiley Periodicals, Inc. PMID:27219203

  4. An Optical Cryostat for Use in Microscopy Cooled by Stirling-Type Pulse Tube Cryocooler

    NASA Astrophysics Data System (ADS)

    Liubiao, Chen; Qiang, Zhou; Xiaoshuang, Zhu; Yuan, Zhou; Junjie, Wang

    The few products of an optical cryostat for use in microscopy in commercialapplications are generally cooled by liquid nitrogen, liquid helium or cryocoolers such as G-M cryocooler or G-M type pulse tube cryocooler (PTC). Sometimes it is not convenient to use G-M cryocooler or G-M type PTC because of its noise and big size; and in some places, liquid nitrogen, especially liquid helium, is not easily available. To overcome this limitation, an optical cryostat for use in microscopy cooled by a Stirling-type pulse tube cryocooler (SPTC) has been designed, built and tested. The refrigerator system SPTC is an important component of the optical cryostat; it has the advantages of compactness, high efficiency, and low vibration. For simplification and compactness, single-stage configuration with coaxial arrangement was employed in the developed SPTC. In order to lower the vibration, the separated configuration was adopted; its compressor and pulse tube are connected with a flexible connecting tube. At present, a lowest temperature of 20 K could be achieved. The temperature fluctuation can be controlled at ±10 mK by adjusting the input electric power to the compressor; and some considerations for further improvement will also be described in this paper.

  5. Fault localization and analysis in semiconductor devices with optical-feedback infrared confocal microscopy

    SciTech Connect

    Sarmiento, Raymund; Cemine, Vernon Julius; Tagaca, Imee Rose; Salvador, Arnel; Mar Blanca, Carlo; Saloma, Caesar

    2007-11-01

    We report on a cost-effective optical setup for characterizing light-emitting semiconductor devices with optical-feedback confocal infrared microscopy and optical beam-induced resistance change.We utilize the focused beam from an infrared laser diode to induce local thermal resistance changes across the surface of a biased integrated circuit (IC) sample. Variations in the multiple current paths are mapped by scanning the IC across the focused beam. The high-contrast current maps allow accurate differentiation of the functional and defective sites, or the isolation of the surface-emittingp-i-n devices in the IC. Optical beam-induced current (OBIC) is not generated since the incident beam energy is lower than the bandgap energy of the p-i-n device. Inhomogeneous current distributions in the IC become apparent without the strong OBIC background. They are located at a diffraction-limited resolution by referencing the current maps against the confocal reflectance image that is simultaneously acquired via optical-feedback detection. Our technique permits the accurate identification of metal and semiconductor sites as well as the classification of different metallic structures according to thickness, composition, or spatial inhomogeneity.

  6. Stochastic Optical Reconstruction Microscopy Imaging of Microtubule Arrays in Intact Arabidopsis thaliana Seedling Roots

    PubMed Central

    Dong, Bin; Yang, Xiaochen; Zhu, Shaobin; Bassham, Diane C.; Fang, Ning

    2015-01-01

    Super-resolution fluorescence microscopy has generated tremendous success in revealing detailed subcellular structures in animal cells. However, its application to plant cell biology remains extremely limited due to numerous technical challenges, including the generally high fluorescence background of plant cells and the presence of the cell wall. In the current study, stochastic optical reconstruction microscopy (STORM) imaging of intact Arabidopsis thaliana seedling roots with a spatial resolution of 20–40 nm was demonstrated. Using the super-resolution images, the spatial organization of cortical microtubules in different parts of a whole Arabidopsis root tip was analyzed quantitatively, and the results show the dramatic differences in the density and spatial organization of cortical microtubules in cells of different differentiation stages or types. The method developed can be applied to plant cell biological processes, including imaging of additional elements of the cytoskeleton, organelle substructure, and membrane domains. PMID:26503365

  7. Evaluation of fractional photothermolysis effect in a mouse model using nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Guo, Han Wen; Tseng, Te-Yu; Dong, Chen-Yuan; Tsai, Tsung-Hua

    2014-07-01

    Fractional photothermolysis (FP) induces discrete columns of photothermal damage in skin dermis, thereby promoting collagen regeneration. This technique has been widely used for treating wrinkles, sun damage, and scar. In this study, we evaluate the potential of multiphoton microscopy as a noninvasive imaging modality for the monitoring of skin rejuvenation following FP treatment. The dorsal skin of a nude mouse underwent FP treatment in order to induce microthermal zones (MTZs). We evaluated the effect of FP on skin remodeling at 7 and 14 days after treatment. Corresponding histology was performed for comparison. After 14 days of FP treatment at 10 mJ, the second harmonic generation signal recovered faster than the skin treated with 30 mJ, indicating a more rapid regeneration of dermal collagen at 10 mJ. Our results indicate that nonlinear optical microscopy is effective in detecting the damaged areas of MTZ and monitoring collagen regeneration following FP treatment.

  8. In vivo corneal neovascularization imaging by optical-resolution photoacoustic microscopy

    PubMed Central

    Liu, Wenzhong; Schultz, Kathryn M.; Zhang, Kevin; Sasman, Amy; Gao, Fengli; Kume, Tsutomu; Zhang, Hao F.

    2014-01-01

    Corneal neovascularization leads to blurred vision, thus in vivo visualization is essential for pathological studies in animal models. Photoacoustic (PA) imaging can delineate microvasculature and hemodynamics noninvasively, which is suitable for investigating corneal neovascularization. In this study, we demonstrate in vivo imaging of corneal neovascularization in the mouse eye by optical-resolution photoacoustic microscopy (OR-PAM), where corneal neovascularization is induced by deliberate alkali burn injuries in C57BL6/J inbred mice corneas on the left eye. We used OR-PAM to image five mice with corneal alkali burn injuries; the uninjured eyes (right eye) in these mice are then used as the controls. Corneal images acquired by OR-PAM with and without alkali burn injury are compared, clear signs of corneal neovascularization are present in the OR-PAM images of injured eyes; the OR-PAM results are also confirmed by postmortem fluorescence-labeled confocal microscopy. PMID:25013754

  9. Utilizing nonlinear optical microscopy to investigate the development of early cancer in nude mice in vivo

    NASA Astrophysics Data System (ADS)

    Wang, Chun-Chin; Li, Feng-Chieh; Lin, Sung-Jan; Lo, Wen; Dong, Chen-Yuan

    2007-07-01

    In this investigation, we used in vivo nonlinear optical microscopy to image normal and carcinogen DMBA treated skin tissues of nude mice. We acquired two-photon autofluroescence and second harmonic generation (SHG) images of the skin tissue, and applied the ASI (Autofluorescence versus SHG Index) to the resulting image. This allows us to visualize and quantify the interaction between mouse skin cells and the surrounding connective tissue. We found that as the imaging depth increases, ASI has a different distribution in the normal and the treated skin tissues. Since the DMBA treated skin eventually became squamous cell carcinoma (SCC), our results show that the physiological changes to mouse skin en route to become cancer can be effectively tracked by multiphoton microscopy. We envision this approach to be effective in studying tumor biology and tumor treatment procedures.

  10. Rapid Discrimination of Polymorphic Crystal Forms by Nonlinear Optical Stokes Ellipsometric Microscopy.

    PubMed

    Schmitt, Paul D; DeWalt, Emma L; Dow, Ximeng Y; Simpson, Garth J

    2016-06-01

    The use of nonlinear optical Stokes ellipsometric (NOSE) microscopy for rapid discrimination of two polymorphic forms of the small molecule d-mannitol is presented. Fast (8 MHz) polarization modulated beam-scanning microscopy and a recently developed iterative, nonlinear least-squares fitting algorithm were combined to allow discrimination of orthorhombic and monoclinic crystal structures of d-mannitol with data acquisition times of <7 s per field of view with a signal-to-noise ratio (SNR) of ∼300. Discrimination between polymorphic forms within the 99.99% confidence interval was achieved by standard statistical tests of the recovered probability density functions for the measured observables following two class linear discriminant analysis. These measurements target bottlenecks in small-volume, high-throughput solid form screening experiments for polymorph discovery in the development of emerging active pharmaceutical ingredients. PMID:27092390

  11. High resolution double-sided diffractive optics for hard X-ray microscopy.

    PubMed

    Mohacsi, Istvan; Vartiainen, Ismo; Guizar-Sicairos, Manuel; Karvinen, Petri; Guzenko, Vitaliy A; Müller, Elisabeth; Färm, Elina; Ritala, Mikko; Kewish, Cameron M; Somogyi, Andrea; David, Christian

    2015-01-26

    The fabrication of high aspect ratio metallic nanostructures is crucial for the production of efficient diffractive X-ray optics in the hard X-ray range. We present a novel method to increase their structure height via the double-sided patterning of the support membrane. In transmission, the two Fresnel zone plates on the two sides of the substrate will act as a single zone plate with added structure height. The presented double-sided zone plates with 30 nm smallest zone width offer up to 9.9% focusing efficiency at 9 keV, that results in a factor of two improvement over their previously demonstrated single-sided counterparts. The increase in efficiency paves the way to speed up X-ray microscopy measurements and allows the more efficient utilization of the flux in full-field X-ray microscopy. PMID:25835837

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

    NASA Astrophysics Data System (ADS)

    Krzewina, Leo G.; Kim, Myung K.

    2006-02-01

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

  13. Nonlinear optical spectroscopy and microscopy of model random and biological media

    NASA Astrophysics Data System (ADS)

    Guo, Yici

    Nonlinear optical (NLO) spectroscopy and microscopy applied to biomedical science are emerging as new and rapidly growing areas which offer important insight into basic phenomena. Ultrafast NLO processes provide temporal, spectral and spatial sensitivities complementary or superior to those achieved through conventional linear optical approaches. The goal of this thesis is to explore the potential of two fundamental NLO processes to produce noninvasive histological maps of biological tissues. Within the goal of the thesis, steady state intensity, polarization and angular measurements of second- and third-harmonic generations (SHG, THG) have been performed on model random scattering and animal tissue samples. The nonlinear optical effects have been evaluated using models. Conversion efficiencies of SHG and THG from animal tissue interfaces have been determined, ranging from 10-7 to 10-10. The changes in the multiharmonic signals were found to depend on both local and overall histological structures of biological samples. The spectral signatures of two photon excitation induced fluorescence from intrinsic fluorophores have been acquired and used to characterize the physical state and types of tissues. Two dimensional scanning SHG and TPF tomographic images have been obtained from in vitro animal tissues, normal and diseased human breast tissues, and resolved subsurface layers and histo-chemical distributions. By combining consecutive 2D maps, a 3D image can be produced. The structure and morphology dependence of the SH signal has been utilized to image and evaluate subsurface tumor progression depth. Second harmonic microscopy in model random and biological cells has been studied using a CCD camera to obtain direct images from subcellular structures. Finally, near infrared (NIR) NLO spectroscopy and microscopy based on SHG and TPF have demonstrated high spatial resolution, deeper penetration depth, low level photo-damaging and enhanced morphological sensitivity for

  14. Nanoscale optical and electrical characterizations of ZnO nanostructures by near-field microscopy

    NASA Astrophysics Data System (ADS)

    Bercu, Bogdan; Giraudet, Louis; Molinari, Michael

    2014-03-01

    The interest in the recent years for nanostructure studies has led to the development of a wide palette of characterization techniques such as the electrical modes in scanning probe microscopy (STM, EFM, KPFM...). Optical characterization at nanoscale remains nevertheless a challenge especially for wide gap semiconductors where high energy is required. In this presentation, we will present our work focusing in the development and the improvement of near-field microscopy techniques to investigate nanoscale properties of ZnO nanostructures and related semiconducting objects. For the optical characterization, cathodoluminescence (CL) studies present many advantages over the classical photoluminescence experiments for ZnO analysis. This contribution presents the development of a scanning near-field cathodoluminescence microscope where a bimorph piezoelectric cantilever is simultaneously used for both actuation and oscillation amplitude detection. Operated inside a scanning electron microscope (SEM) it offers the possibility of performing simultaneous topography and cathodoluminescence charting of the sample surface additionally to the SEM imaging with a resolution in the order of several tenths of nanometers. Different measurements of ZnO nanostructures and related objects will be presented to show the potentiality of our optical characterization setup. Complementary STEM-CL measurements at higher beam energy were performed on the ZnO nanowires confirming the good quality of the investigated nanostructures. As for the electrical characterization, we will focus on the local surface potential mapping of ZnO nanowires used for photoconduction using Kelvin Probe Force Microscopy. While ZnO nanowire photoconduction gains as high as 1010 in the UV region were reported, several issues come into play when it comes to making a precise measurement of a single nanowire. An important issue is the good quality of the injecting contacts on the nanowire and the reproducibility of its

  15. Observation of mesenteric microcirculatory disturbance in rat by laser oblique scanning optical microscopy

    NASA Astrophysics Data System (ADS)

    Ding, Yichen; Zhang, Yu; Peng, Tong; Lu, Yiqing; Jin, Dayong; Ren, Qiushi; Liu, Yuying; Han, Jingyan; Xi, Peng

    2013-05-01

    Ischemia-reperfusion (I/R) injury model has been widely applied to the study of microcirculation disturbance. In this work, we used laser oblique scanning optical microscopy (LOSOM) to observe the microcirculation system in the mesentery of rat model. Utilizing a localized point-scanning detection scheme, high-contrast images of leukocytes were obtained. The extended detection capability facilitated both the automatic in vivo cell counting and the accurate measurement of the rolling velocity of leukocytes. Statistical analysis of the different treatment groups suggested that the distinction between I/R and sham groups with time lapse is significant.

  16. Selective observation of starch in a water plant using optical sum-frequency microscopy

    NASA Astrophysics Data System (ADS)

    Miyauchi, Yoshihiro; Sano, Haruyuki; Mirzutani, Goro

    2006-07-01

    The photosynthesis, transfer, and storage of starch are the most important biogenic processes occurring in plants. In order to observe the colorless and transparent starch granules in a plant, a chemical pretreatment such as staining of the starch is currently required, which seriously damages the tissue cells in the plant. We demonstrate that nondestructive chemical analysis of starch granules in a plant can be performed by using optical second-harmonic and sum-frequency microscopy. These techniques for in vivo analysis will provide extremely useful information about saccharides in a plant and can be extended to the analysis of many other materials, from living tissue to semiconductors.

  17. What advances in microscopy are required for combined MRI and optical functional brain imaging? (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Kleinfeld, David

    2016-03-01

    This overview talk will focus on forward-looking scientific needs and physical limits to images of neuronal processes. The challenge in nervous systems is that the basic unit for "switching" events in the nervous system occurs on the one micrometer scale of synaptic spines, while computations involve communication between individual neurons across the full expanse of cortex, which is ten millimeters for mouse cortex. I will address hoped-for advances in optical microscopy, within the context of existing and proposed contrast mechanisms of neuronal function, that span the four orders of magnitude of length scales for neuronal processing

  18. Optical clearing assisted confocal microscopy of ex vivo transgenic mouse skin

    NASA Astrophysics Data System (ADS)

    Song, Eunjoo; Ahn, YoonJoon; Ahn, Jinhyo; Ahn, Soyeon; Kim, Changhwan; Choi, Sanghoon; Boutilier, Richard Martin; Lee, Yongjoong; Kim, Pilhan; Lee, Ho

    2015-10-01

    We examined the optical clearing assisted confocal microscopy of the transgenic mouse skin. The pinna and dorsal skin were imaged with a confocal microscope after the application of glycerol and FocusClear. In case of the glycerol-treated pinna, the clearing was minimal due to the inefficient permeability. However, the imaging depth was improved when the pinna was treated with FocusClear. In case of dorsal skin, we were able to image deeply to the subcutaneous connective tissue with both agents. Various skin structures such as the vessel, epithelium cells, cartilage, dermal cells, and hair follicles were clearly imaged.

  19. Approximate Bayesian computation for estimating number concentrations of monodisperse nanoparticles in suspension by optical microscopy

    NASA Astrophysics Data System (ADS)

    Röding, Magnus; Zagato, Elisa; Remaut, Katrien; Braeckmans, Kevin

    2016-06-01

    We present an approximate Bayesian computation scheme for estimating number concentrations of monodisperse diffusing nanoparticles in suspension by optical particle tracking microscopy. The method is based on the probability distribution of the time spent by a particle inside a detection region. We validate the method on suspensions of well-controlled reference particles. We illustrate its usefulness with an application in gene therapy, applying the method to estimate number concentrations of plasmid DNA molecules and the average number of DNA molecules complexed with liposomal drug delivery particles.

  20. Phase stabilized homodyne of infrared scattering type scanning near-field optical microscopy

    SciTech Connect

    Xu, Xiaoji G.; Gilburd, Leonid; Walker, Gilbert C.

    2014-12-29

    Scattering type scanning near-field optical microscopy (s-SNOM) allows sub diffraction limited spatial resolution. Interferometric homodyne detection in s-SNOM can amplify the signal and extract vibrational responses based on sample absorption. A stable reference phase is required for a high quality homodyne-detected near-field signal. This work presents the development of a phase stabilization mechanism for s-SNOM to provide stable homodyne conditions. The phase stability is found to be better than 0.05 rad for the mid infrared light source. Phase stabilization results in improved near field images and vibrational spectroscopies. Spatial inhomogeneities of the boron nitride nanotubes are measured and compared.

  1. Observation of mesenteric microcirculatory disturbance in rat by laser oblique scanning optical microscopy

    PubMed Central

    Ding, Yichen; Zhang, Yu; Peng, Tong; Lu, Yiqing; Jin, Dayong; Ren, Qiushi; Liu, Yuying; Han, Jingyan; Xi, Peng

    2013-01-01

    Ischemia-reperfusion (I/R) injury model has been widely applied to the study of microcirculation disturbance. In this work, we used laser oblique scanning optical microscopy (LOSOM) to observe the microcirculation system in the mesentery of rat model. Utilizing a localized point-scanning detection scheme, high-contrast images of leukocytes were obtained. The extended detection capability facilitated both the automatic in vivo cell counting and the accurate measurement of the rolling velocity of leukocytes. Statistical analysis of the different treatment groups suggested that the distinction between I/R and sham groups with time lapse is significant. PMID:23640310

  2. In vivo noninvasive monitoring of microhemodynamics using optical-resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Hu, Song; Maslov, Konstantin I.; Wang, Lihong V.

    2009-02-01

    Microvascular autoregulation is an intrinsic ability of vascular beds to compensate for the fluctuation in blood flow and tissue oxygen delivery. This function is crucial to maintaining the local metabolic activity. Here, using optical-resolution photoacoustic microscopy (OR-PAM), we clearly observed vasomotion and vasodilation in the intact mouse microcirculation in vivo in response to the changes in physiological state. Our results show that a significant lowfrequency vasomotion can be seen under hyperoxia but not hypoxia. Moreover, significant vasodilation is observed when the animal status is switched from hyperoxia to hypoxia. Our data show that arterioles have more pronounced vasodilation than venules.

  3. Alterations of single molecule fluorescence lifetimes in near-field optical microscopy

    SciTech Connect

    Ambrose, W.P.; Goodwin, P.M.; Keller, R.A.; Martin, J.C. )

    1994-07-15

    Fluorescence lifetimes of single Rhodamine 6G molecules on silica surfaces were measured with pulsed laser excitation, time-correlated single photon counting, and near-field scanning optical microscopy (NSOM). The fluorescence lifetime varies with the position of a molecule relative to a near-field probe. Qualitative features of lifetime decreases are consistent with molecular excited state quenching effects near metal surfaces. The technique of NSOM provides a means of altering the environment of a single fluorescent molecule and its decay kinetics in a repeatable fashion.

  4. Occlusal overload investigations by noninvasive technology: fluorescence microscopy and en-face optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Marcauteanu, Corina; Negrutiu, Meda; Sinescu, Cosmin; Demjan, Enikö; Hughes, Michael; Bradu, Adrian; Dobre, George; Podoleanu, Adrian G.

    2009-07-01

    The aim of this study is the early detection and monitoring of occlusal overload in bruxing patients. En-Face Optical coherence tomography (eF-OCT) and fluorescence microscopy (FM) were used for the imaging of several anterior teeth extracted from patients with light active bruxism. We found a characteristic pattern of enamel cracks, that reached the tooth surface. We concluded that the combination of the en-Face OCT and FM is a promising non-invasive alternative technique for reliable monitoring of occlusal overload.

  5. Fast optical sectioning obtained by structured illumination microscopy using a digital mirror device

    NASA Astrophysics Data System (ADS)

    Xu, Dongli; Jiang, Tao; Li, Anan; Hu, Bihe; Feng, Zhao; Gong, Hui; Zeng, Shaoqun; Luo, Qingming

    2013-06-01

    High-throughput optical imaging is critical to obtain large-scale neural connectivity information of brain in neuroscience. Using a digital mirror device and a scientific complementary metal-oxide semiconductor camera, we report a significant speed improvement of structured illumination microscopy (SIM), which produces a maximum SIM net frame rate of 133 Hz. We perform three-dimensional (3-D) imaging of mouse brain slices at diffraction-limited resolution and demonstrate the fast 3-D imaging capability to a large sample with an imaging rate of 6.9 pixel/s of our system, an order of magnitude faster than previously reported.

  6. Study of environmental biodegradation of LDPE films in soil using optical and scanning electron microscopy.

    PubMed

    Mumtaz, Tabassum; Khan, M R; Hassan, Mohd Ali

    2010-07-01

    An outdoor soil burial test was carried out to evaluate the degradation of commercially available LDPE carrier bags in natural soil for up to 2 years. Biodegradability of low density polyethylene films in soil was monitored using both optical and scanning electron microscopy (SEM). After 7-9 months of soil exposure, microbial colonization was evident on the film surface. Exposed LDPE samples exhibit progressive changes towards degradation after 17-22 months. SEM images reveal signs of degradation such as exfoliation and formation of cracks on film leading to disintegration. The possible degradation mode and consequences on the use and disposal of LDPE films is discussed. PMID:20207547

  7. Simultaneous optical coherence tomography and autofluorescence microscopy with a single light source

    NASA Astrophysics Data System (ADS)

    Dai, Cuixia; Liu, Xiaojing; Jiao, Shuliang

    2012-08-01

    We have accomplished simultaneous spectral domain optical coherence tomography (SD-OCT) and autofluorescence (AF) microscopy with a broadband light source centered at 415 nm. The light source was provided by frequency-doubling of an ultra-fast broadband Ti:Sapphire laser. With a bandwidth of 8 nm, the visible SD-OCT achieved a depth resolution of ˜12 μm. Since the two imaging modalities are provided by the same group of photons, their images are intrinsically registered. The dual-modal system is capable of providing OCT imaging and molecular contrasts simultaneously. The imaging system was tested on imaging biological samples ex vivo and in vivo.

  8. In vivo optical microscopy of peripheral nerve myelination with polarization sensitive-optical coherence tomography.

    PubMed

    Henry, Francis P; Wang, Yan; Rodriguez, Carissa L R; Randolph, Mark A; Rust, Esther A Z; Winograd, Jonathan M; de Boer, Johannes F; Park, B Hyle

    2015-04-01

    Assessing nerve integrity and myelination after injury is necessary to provide insight for treatment strategies aimed at restoring neuromuscular function. Currently, this is largely done with electrical analysis, which lacks direct quantitative information. In vivo optical imaging with sufficient imaging depth and resolution could be used to assess the nerve microarchitecture. In this study, we examine the use of polarization sensitive-optical coherence tomography (PS-OCT) to quantitatively assess the sciatic nerve microenvironment through measurements of birefringence after applying a nerve crush injury in a rat model. Initial loss of function and subsequent recovery were demonstrated by calculating the sciatic function index (SFI). We found that the PS-OCT phase retardation slope, which is proportional to birefringence, increased monotonically with the SFI. Additionally, histomorphometric analysis of the myelin thickness and g-ratio shows that the PS-OCT slope is a good indicator of myelin health and recovery after injury. These results demonstrate that PS-OCT is capable of providing nondestructive and quantitative assessment of nerve health after injury and shows promise for continued use both clinically and experimentally in neuroscience. PMID:25858593

  9. In vivo optical microscopy of peripheral nerve myelination with polarization sensitive-optical coherence tomography

    PubMed Central

    Henry, Francis P.; Wang, Yan; Rodriguez, Carissa L. R.; Randolph, Mark A.; Rust, Esther A. Z.; Winograd, Jonathan M.; de Boer, Johannes F.; Park, B. Hyle

    2015-01-01

    Abstract. Assessing nerve integrity and myelination after injury is necessary to provide insight for treatment strategies aimed at restoring neuromuscular function. Currently, this is largely done with electrical analysis, which lacks direct quantitative information. In vivo optical imaging with sufficient imaging depth and resolution could be used to assess the nerve microarchitecture. In this study, we examine the use of polarization sensitive-optical coherence tomography (PS-OCT) to quantitatively assess the sciatic nerve microenvironment through measurements of birefringence after applying a nerve crush injury in a rat model. Initial loss of function and subsequent recovery were demonstrated by calculating the sciatic function index (SFI). We found that the PS-OCT phase retardation slope, which is proportional to birefringence, increased monotonically with the SFI. Additionally, histomorphometric analysis of the myelin thickness and g-ratio shows that the PS-OCT slope is a good indicator of myelin health and recovery after injury. These results demonstrate that PS-OCT is capable of providing nondestructive and quantitative assessment of nerve health after injury and shows promise for continued use both clinically and experimentally in neuroscience. PMID:25858593

  10. Nonlinear optical Stokes ellipsometric (NOSE) microscopy for imaging the nonlinear susceptibility tensors of collagen

    NASA Astrophysics Data System (ADS)

    Dow, Ximeng Y.; DeWalt, Emma L.; Sullivan, Shane Z.; Schmitt, Paul D.; Simpson, Garth J.

    2016-03-01

    Nonlinear optical Stokes ellipsometric (NOSE) microscopy was demonstrated for the analysis of collagen structure in a mouse tail section. NOSE is based on polarization-dependent second harmonic generation (SHG) imaging. The fast polarization-modulation was achieved using an electro-optic modulator (EOM), allowing for the potential of video-rate NOSE analysis. The signal to noise advantages associated with suppression of 1/f noise by rapid polarization modulation allowed reliable recovery of the local-frame tensor on a per-pixel basis. An iterative approach involving laboratory to local frame coordinate transformation was developed to recover the spatial distribution of local-frame nonlinear susceptibility tensor elements of collagen as well as the polar and azimuthal orientation angles of the collagen structure.

  11. Combined photoacoustic microscopy and optical coherence tomography can measure metabolic rate of oxygen

    PubMed Central

    Liu, Tan; Wei, Qing; Wang, Jing; Jiao, Shuliang; Zhang, Hao F.

    2011-01-01

    We proposed to measure the metabolic rate of oxygen (MRO2) in small animals in vivo using a multimodal imaging system that combines laser-scanning optical-resolution photoacoustic microscopy (LSOR-PAM) and spectral-domain optical coherence tomography (SD-OCT). We first tested the capability of the multimodal system to measure flow rate in a phantom made of two capillary tubes of different diameters. We then demonstrated the capability of measuring MRO2 by imaging two parallel vessels selected from the ear of a Swiss Webster mouse. The hemoglobin oxygen saturation (sO2) and the vessel diameter were measured by the LSOR-PAM and the blood flow velocity was measured by the SD-OCT, from which blood flow rate and MRO2 were further calculated. The measured blood flow rates in the two vessels agreed with each other. PMID:21559147

  12. Mapping electron-beam-injected trapped charge with scattering scanning near-field optical microscopy.

    PubMed

    Tranca, Denis E; Sánchez-Ortiga, Emilio; Saavedra, Genaro; Martínez-Corral, Manuel; Tofail, Syed A M; Stanciu, Stefan G; Hristu, Radu; Stanciu, George A

    2016-03-01

    Scattering scanning near-field optical microscopy (s-SNOM) has been demonstrated as a valuable tool for mapping the optical and optoelectronic properties of materials with nanoscale resolution. Here we report experimental evidence that trapped electric charges injected by an electron beam at the surface of dielectric samples affect the sample-dipole interaction, which has direct impact on the s-SNOM image content. Nanoscale mapping of the surface trapped charge holds significant potential for the precise tailoring of the electrostatic properties of dielectric and semiconductive samples, such as hydroxyapatite, which has particular importance with respect to biomedical applications. The methodology developed here is highly relevant to semiconductor device fabrication as well. PMID:26974112

  13. Spectral-domain optical coherence phase microscopy for label-free multiplexed protein microarray assay

    PubMed Central

    Joo, Chulmin; Özkumur, Emre; Ünlü, M. Selim; de Boer, Johannes F.

    2009-01-01

    Quantitative measurement of affinities and kinetics of various biomolecular interactions such as protein-protein, protein-DNA and receptor-ligand is central to our understanding of basic molecular and cellular functions and is useful for therapeutic evaluation. Here, we describe a laser-scanning quantitative imaging method, referred to as spectral-domain optical coherence phase microscopy, as an optical platform for label-free detection of biomolecular interactions. The instrument is based on a confocal interferometric microscope that enables depth-resolved quantitative phase measurements on sensor surface with high spatial resolution and phase stability. We demonstrate picogram per square millimeter surface mass sensitivity, and show its sensing capability by presenting static and dynamic detection of multiplexed protein microarray as immobilized antigens capture their corresponding antibodies. PMID:19674885

  14. In-vivo monitoring rat skin wound healing using nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Jing; Guo, Chungen; Zhang, Fan; Xu, Yahao; Zhu, Xiaoqin; Xiong, Shuyuan; Chen, Jianxin

    2014-11-01

    Nonlinear optical microscopy (NLOM) was employed for imaging and evaluating the wound healing process on rat skin in vivo. From the high-resolution nonlinear optical images, the morphology and distribution of specific biological markers in cutaneous wound healing such as fibrin clot, collagens, blood capillaries, and hairs were clearly observed at 1, 5 and 14 days post injury. We found that the disordered collagen in the fibrin clot at day 1 was replaced by regenerative collagen at day 5. By day 14, the thick collagen with well-network appeared at the original margin of the wound. These findings suggested that NLOM is ideal for noninvasively monitoring the progress of wound healing in vivo.

  15. Near-field optical microscopy and spectroscopy of few-layer black phosphorous

    NASA Astrophysics Data System (ADS)

    Frenzel, A. J.; Tran, S.; Hinton, J. P.; Sternbach, A. J.; Yang, J.; Gillgren, N.; Lau, C. N.; Basov, D. N.

    Few-layer black phosphorous is a recent addition to the family of two-dimensional (2D) materials which exhibits strongly anisotropic transport and optical properties due to its puckered honeycomb structure. It was recently predicted that this intrinsic anisotropy should manifest in the plasmon dispersion. Additionally, tuning layer number and carrier density can control the dispersion of these collective modes. Scanning near-field optical microscopy (SNOM) has been demonstrated as a powerful method to probe electronic properties, including propagating collective modes, in layered 2D materials. We used SNOM to investigate anisotropic carrier response in few-layer black phosphorous encapsulated by hexagonal boron nitride. In addition to exploring gate-voltage tunability of the electronic response, we demonstrate effective modulation of the near-field signal by ultrafast photoexcitation.

  16. Imaging and graphing of cortical vasculature using dynamically focused optical coherence microscopy angiography

    NASA Astrophysics Data System (ADS)

    Leahy, Conor; Radhakrishnan, Harsha; Bernucci, Marcel; Srinivasan, Vivek J.

    2016-02-01

    Recently, optical coherence tomography (OCT) angiography has enabled label-free imaging of vasculature based on dynamic scattering in vessels. However, quantitative volumetric analysis of the vascular networks depicted in OCT angiography data has remained challenging. Multiple-scattering tails (artifacts specific to the imaging geometry) make automated assessment of vascular morphology problematic. We demonstrate that dynamically focused optical coherence microscopy (OCM) angiography with a high numerical aperture, chosen so the scattering length greatly exceeds the depth-of-field, significantly reduces the deleterious effect of multiple-scattering tails in synthesized angiograms. Capitalizing on the improved vascular image quality, we devised and tailored a self-correcting automated graphing approach that achieves a reconstruction of cortical microvasculature from OCM angiography data sets with accuracy approaching that attained by trained operators. The automated techniques described here will facilitate more widespread study of vascular network topology in health and disease.

  17. Femtosecond infrared intrastromal ablation and backscattering-mode adaptive-optics multiphoton microscopy in chicken corneas

    PubMed Central

    Gualda, Emilio J.; Vázquez de Aldana, Javier R.; Martínez-García, M. Carmen; Moreno, Pablo; Hernández-Toro, Juan; Roso, Luis; Artal, Pablo; Bueno, Juan M.

    2011-01-01

    The performance of femtosecond (fs) laser intrastromal ablation was evaluated with backscattering-mode adaptive-optics multiphoton microscopy in ex vivo chicken corneas. The pulse energy of the fs source used for ablation was set to generate two different ablation patterns within the corneal stroma at a certain depth. Intrastromal patterns were imaged with a custom adaptive-optics multiphoton microscope to determine the accuracy of the procedure and verify the outcomes. This study demonstrates the potential of using fs pulses as surgical and monitoring techniques to systematically investigate intratissue ablation. Further refinement of the experimental system by combining both functions into a single fs laser system would be the basis to establish new techniques capable of monitoring corneal surgery without labeling in real-time. Since the backscattering configuration has also been optimized, future in vivo implementations would also be of interest in clinical environments involving corneal ablation procedures. PMID:22076258

  18. Double-resonance probe for near-field scanning optical microscopy

    NASA Astrophysics Data System (ADS)

    Cherkun, A. P.; Serebryakov, D. V.; Sekatskii, S. K.; Morozov, I. V.; Letokhov, V. S.

    2006-03-01

    A surface-contact transducer is developed for scanning probe microscopes, whose operating principle is based on the coincidence between the resonance frequency of a 32kHz quartz tuning fork and that of the probe attached to it. This allows the transducer to have a high quality factor and, if the vibration amplitude of the probe tip exceeds that of the tuning fork prongs, materially improves its force sensitivity. The resonance transducer proposed by us has an experimentally verified force sensitivity of 8pN (rms) in the 300Hz frequency band, which is of the same order of magnitude as the sensitivity of atomic force microscope (AFM) cantilever sensors. The manufacture of such transducers equipped with optical-fiber probes for near-field scanning optical microscopy and with tungsten probes for AFM is described as an example.

  19. Massively parallel processor networks with optical express channels

    DOEpatents

    Deri, R.J.; Brooks, E.D. III; Haigh, R.E.; DeGroot, A.J.

    1999-08-24

    An optical method for separating and routing local and express channel data comprises interconnecting the nodes in a network with fiber optic cables. A single fiber optic cable carries both express channel traffic and local channel traffic, e.g., in a massively parallel processor (MPP) network. Express channel traffic is placed on, or filtered from, the fiber optic cable at a light frequency or a color different from that of the local channel traffic. The express channel traffic is thus placed on a light carrier that skips over the local intermediate nodes one-by-one by reflecting off of selective mirrors placed at each local node. The local-channel-traffic light carriers pass through the selective mirrors and are not reflected. A single fiber optic cable can thus be threaded throughout a three-dimensional matrix of nodes with the x,y,z directions of propagation encoded by the color of the respective light carriers for both local and express channel traffic. Thus frequency division multiple access is used to hierarchically separate the local and express channels to eliminate the bucket brigade latencies that would otherwise result if the express traffic had to hop between every local node to reach its ultimate destination. 3 figs.

  20. Massively parallel processor networks with optical express channels

    DOEpatents

    Deri, Robert J.; Brooks, III, Eugene D.; Haigh, Ronald E.; DeGroot, Anthony J.

    1999-01-01

    An optical method for separating and routing local and express channel data comprises interconnecting the nodes in a network with fiber optic cables. A single fiber optic cable carries both express channel traffic and local channel traffic, e.g., in a massively parallel processor (MPP) network. Express channel traffic is placed on, or filtered from, the fiber optic cable at a light frequency or a color different from that of the local channel traffic. The express channel traffic is thus placed on a light carrier that skips over the local intermediate nodes one-by-one by reflecting off of selective mirrors placed at each local node. The local-channel-traffic light carriers pass through the selective mirrors and are not reflected. A single fiber optic cable can thus be threaded throughout a three-dimensional matrix of nodes with the x,y,z directions of propagation encoded by the color of the respective light carriers for both local and express channel traffic. Thus frequency division multiple access is used to hierarchically separate the local and express channels to eliminate the bucket brigade latencies that would otherwise result if the express traffic had to hop between every local node to reach its ultimate destination.

  1. An ultra-low noise optical head for liquid environment atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Schlesinger, I.; Kuchuk, K.; Sivan, U.

    2015-08-01

    The design considerations and eventual performance of a new, ultra-low noise optical head for dynamic atomic force microscopy (AFM) are presented. The head, designed specifically for the study of hydration layers and ion organization next to solid surfaces and biomolecules, displays an integrated tip-sample distance noise below 3 pm. The sensitivity of the optical beam deflection sensor, operating at frequencies up to 8.6 MHz (3 dB roll-off), is typically below 10 fm / √{ Hz } , enabling utilization of high frequency cantilevers of low thermal noise for fundamental and higher mode imaging. Exceptional signal stability and low optical noise are achieved by replacing the commonly used laser diode with a helium-neon laser. An integral photothermal excitation of the cantilever produces pure harmonic oscillations, minimizing the generation of higher cantilever modes and deleterious sound waves characterizing the commonly used excitation by a piezoelectric crystal. The optical head is designed to fit on top of the widespread Multimode® (Bruker) piezo-tube and accommodate its commercial liquid cell. The performance of the new AFM head is demonstrated by atomic resolution imaging of a muscovite mica surface in aqueous solution.

  2. An ultra-low noise optical head for liquid environment atomic force microscopy.

    PubMed

    Schlesinger, I; Kuchuk, K; Sivan, U

    2015-08-01

    The design considerations and eventual performance of a new, ultra-low noise optical head for dynamic atomic force microscopy (AFM) are presented. The head, designed specifically for the study of hydration layers and ion organization next to solid surfaces and biomolecules, displays an integrated tip-sample distance noise below 3 pm. The sensitivity of the optical beam deflection sensor, operating at frequencies up to 8.6 MHz (3 dB roll-off), is typically below 10 fm/√Hz, enabling utilization of high frequency cantilevers of low thermal noise for fundamental and higher mode imaging. Exceptional signal stability and low optical noise are achieved by replacing the commonly used laser diode with a helium-neon laser. An integral photothermal excitation of the cantilever produces pure harmonic oscillations, minimizing the generation of higher cantilever modes and deleterious sound waves characterizing the commonly used excitation by a piezoelectric crystal. The optical head is designed to fit on top of the widespread Multimode(®) (Bruker) piezo-tube and accommodate its commercial liquid cell. The performance of the new AFM head is demonstrated by atomic resolution imaging of a muscovite mica surface in aqueous solution. PMID:26329201

  3. Transmission polarized optical microscopy of short-pitch cholesteric liquid crystal shells

    NASA Astrophysics Data System (ADS)

    Geng, Yong; Noh, JungHyun; Lagerwall, Jan P. F.

    2016-03-01

    We recently demonstrated that colloidal crystal arrangements of monodisperse droplets or shells of planar-aligned cholesteric liquid crystal exhibit intricate patterns of circularly polarized reflection spots of different colors. The spots appear as a result of photonic cross communication between droplets, hence the patterns reflect the macroscopic arrangement of droplets or shells. Apart from being an interesting optical phenomenon, it offers attractive application opportunities in photonics and beyond, due to the unique characteristics of the patterns. It turns out that the optical quality of shells is much enhanced compared to that of droplets, hence we focus our attention primarily on shells, of varying thickness. Here we analyze and explain the intriguing textures arising when studying planar-aligned short-pitch cholesteric shells in transmission polarizing optical microscopy. In this case, the texture reflects the properties of each individual shell, without any sign of cross communication, yet also this pattern holds some fascinating mysteries. These can only be elucidated by considering all the peculiar optical properties of cholesterics together, as well as the unusual situation given by the spherical shell geometry.

  4. Bimodal endoscopic probe combining white-light microscopy and optical coherence tomography.

    PubMed

    Blattmann, M; Kretschmer, S; Thiele, S; Ataman, C; Zappe, H; Herkommer, A; Seifert, A

    2016-05-20

    We present a novel bimodal endoscopic imaging probe that can simultaneously provide full-field white-light video microscopy and confocal optical coherence tomography (OCT) depth scans. The two modalities rely on spectrally separated optical paths that run partially in parallel through a micro-optical bench system, which has a cross-section of only 2  mm×2.76  mm and is realized via standard silicon micromachining techniques. With a numerical aperture of 0.061, the video modality has a resolution and field of view of 9.3 and 1240  μm×1080  μm, respectively. The resolution is limited by the pixel spacing of the coherent fiber bundle, which relays the acquired image from the distal to the proximal end. A custom-designed diffractive optical element placed within the video imaging path significantly improves the image contrast by up to 45% in the medium frequency range. The OCT modality is optimized for 830 nm center wavelength, and works in a confocal arrangement with an NA of 0.018. It provides single-point depth probing at the center of the video image with a lateral resolution of 20 μm. Through its compact footprint and enhanced functionality, the probe can provide depth-resolved guiding capability for existing laparoscopes and represents a major step toward a new class of multimodal endoscopic imaging probes. PMID:27411158

  5. Correlation between polarization sensitive optical coherence tomography and second harmonic generation microscopy in skin.

    PubMed

    Le, Viet-Hoan; Lee, Seunghun; Kim, Bumju; Yoon, Yeoreum; Yoon, Calvin J; Chung, Wan Kyun; Kim, Ki Hean

    2015-07-01

    Both polarization sensitive optical coherence tomography (PS-OCT) and second harmonic generation (SHG) microscopy are 3D optical imaging methods providing information related to collagen in the skin. PS-OCT provides birefringence information which is due to the collagen composition of the skin. SHG microscopy visualizes collagen fibers in the skin based on their SHG property. These two modalities have been applied to the same skin pathologies associated with collagen changes, but their relationship has not been examined. In this study, we tried to find the relationship by imaging the same skin samples with both modalities. Various parts of the normal rat skin and burn damaged skin were imaged ex vivo, and their images were analyzed both qualitatively and quantitatively. PS-OCT images were analyzed to obtain tissue birefringence. SHG images were analyzed to obtain collagen orientation indices by applying 2D Fourier transform. The skin samples having higher birefringence values had higher collagen orientation indices, and a linear correlation was found between them. Burn damaged skin showed decreases in both parameters compared to the control skins. This relationship between the bulk and microscopic properties of skin may be useful for further skin studies. PMID:26203380

  6. Lensless fluorescent on-chip microscopy using a fiber-optic taper.

    PubMed

    Coskun, Ahmet F; Sencan, Ikbal; Su, Ting-Wei; Ozcan, Aydogan

    2011-01-01

    We demonstrate a lensfree on-chip fluorescent microscopy platform that can image fluorescently labeled cells over ~60 mm(2) field-of-view with <4 urn spatial resolution. In this lensfree imaging system, micro-objects of interest are directly located on a tapered fiber-optic faceplate which has > 5-fold higher density of fiber-optic waveguides in its top facet compared to the bottom facet. For excitation, an incoherent light source (e.g., a simple light emitting diode--LED) is used to pump fluorescent objects through a glass hemi-sphere interface. Upon interacting with the entire sample volume, the excitation light is rejected by total internal reflection process occurring at the bottom of the sample substrate. Fluorescent emission from the objects is then collected by the smaller facet of the tapered faceplate and is delivered to a detector-array with an image magnification of ~2.4X. A compressive sampling based decoding algorithm is used for sparse signal recovery, which further increases the space-bandwidth-product of our lensfree on-chip fluorescent imager. We validated the performance of this lensfree imaging platform using fluorescent micro-particles as well as labeled water-borne parasites (e.g., Giardia Muris cysts). Such a compact and wide-field fluorescent microscopy platform could be valuable for cytometry and rare cell imaging applications as well as for micro array research. PMID:22255702

  7. In Vivo Confocal Microscopy and Anterior Segment Optic Coherence Tomography Findings in Ocular Ochronosis

    PubMed Central

    Demirkilinc Biler, Elif; Guven Yilmaz, Suzan; Palamar, Melis; Hamrah, Pedram

    2015-01-01

    Purpose. To report clinical and in vivo confocal microscopy (IVCM) findings of two patients with ocular ochronosis secondary due to alkaptonuria. Materials and Methods. Complete ophthalmologic examinations, including IVCM (HRT II/Rostock Cornea Module, Heidelberg, Germany), anterior segment optical coherence tomography (AS-OCT) (Topcon 3D spectral-domain OCT 2000, Topcon Medical Systems, Paramus, NJ, USA), corneal topography (Pentacam, OCULUS Optikgeräte GmbH, Wetzlar, Germany), and anterior segment photography, were performed. Results. Biomicroscopic examination showed bilateral darkly pigmented lesions of the nasal and temporal conjunctiva and episclera in both patients. In vivo confocal microscopy of the lesions revealed prominent degenerative changes, including vacuoles and fragmentation of collagen fibers in the affected conjunctival lamina propria and episclera. Hyperreflective pigment granules in different shapes were demonstrated in the substantia propria beneath the basement membrane. AS-OCT of Case 1 demonstrated hyporeflective areas. Fundus examination was within normal limits in both patients, except tilted optic discs with peripapillary atrophy in one of the patients. Corneal topography, thickness, and macular OCT were normal bilaterally in both cases. Conclusion. The degenerative and anatomic changes due to ochronotic pigment deposition in alkaptonuria can be demonstrated in detail with IVCM and AS-OCT. Confocal microscopic analysis in ocular ochronosis may serve as a useful adjunct in diagnosis and monitoring of the disease progression. PMID:26788390

  8. Optical spectroscopy and scanning tunneling microscopy studies of molecular adsorbates and anisotropic ultrathin films. Final report

    SciTech Connect

    Hemminger, J.C.

    1998-09-01

    The bonding, chemistry and ordering of molecular adsorbates on well defined single crystal surfaces and in ultrathin films was to be studied in an effort to develop sufficient fundamental understanding to allow the controlled preparation of anisotropic ultrathin films of organic monolayers. In this research the authors combine the use of optical probes (Raman spectroscopy, laser induced thermal desorption with Fourier transform mass spectrometry detection) with scanning tunneling microscopy (STM) and conventional methods of UHV surface science (Auger electron spectroscopy, x-ray photoelectron spectroscopy, low energy electron diffraction, and thermal desorption spectroscopy). The conventional surface probes provide well tested methods for the preparation and characterization of single crystal substrates. The optical probes used in the experiments provide powerful methods for the molecular identification of adsorbates in monolayers and ultrathin films. Scanning tunneling microscopy provides one with the ability to determine the detailed molecular level ordering of the molecular adsorbates. The emphasis of this research is on more complex molecular absorbates some of which are monomer precursors to ultrathin polymer films. Enhanced methods of Raman spectroscopy have been developed for the study of monolayer adsorbates on surfaces in ultrahigh vacuum environments. This report gives an overview of recent research results, including the construction of UHV variable temperature STM, analysis of STM images, growth and chemistry of intermetallic single crystal ultrathin films, and electron beam induced chemistry of tetracyanoquinodimethane.

  9. A new method of assessing the surgical margin in rectal carcinoma—using nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Li, Lianhuang; Chen, Zhifen; Kang, Deyong; Deng, Tongxin; Jiang, Liwei; Zhou, Yi; Liu, Xing; Jiang, Weizhong; Zhuo, Shuangmu; Guan, Guoxian; Chi, Pan; Chen, Jianxin

    2016-06-01

    Nowadays, surgical resection is still the most effective treatment strategy for rectal carcinoma and one of the most important factors affecting whether the operation is successful or not is the surgical margin determination, especially in the distal rectal carcinoma which should take the sphincter-preserving issue into consideration. However, until recently no reliable evaluation method has been developed for this purpose. There are some shortcomings in intraoperative negative surgical margin assessment such as either lack of enough detailed information of biological tissues or the fact that it is time-consuming. Multiphoton microscopy (MPM)—nonlinear optical microscopy, which is based on the nonlinear optical process two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), has the ability to label freely and noninvasively visualize tissue micro-architecture at the sub-cellular level. The advantage of providing high contrast and high resolution biomedical image in real time makes MPM have a wide range of applications in life sciences. In this study, we introduced MPM to identify the boundary between normal and abnormal rectal tissues. MPM images clearly exhibit biological tissue microstructure and its morphological changes in the regions of our interest, which enable it to determine the surgical margin in rectal carcinoma. It can be foreseen that once MPM imaging system is used in clinical examination, it will greatly improve the accuracy of surgical resection.

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

    NASA Astrophysics Data System (ADS)

    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.

  11. Wide-field lensless fluorescent microscopy using a tapered fiber-optic faceplate on a chip†

    PubMed Central

    Coskun, Ahmet F.; Sencan, Ikbal; Su, Ting-Wei; Ozcan, Aydogan

    2011-01-01

    We demonstrate lensless fluorescent microscopy over a large field-of-view of ~60 mm2 with a spatial resolution of <4 μm. In this on-chip fluorescent imaging modality, the samples are placed on a fiber-optic faceplate that is tapered such that the density of the fiber-optic waveguides on the top facet is >5 fold larger than the bottom one. Placed on this tapered faceplate, the fluorescent samples are pumped from the side through a glass hemisphere interface. After excitation of the samples, the pump light is rejected through total internal reflection that occurs at the bottom facet of the sample substrate. The fluorescent emission from the sample is then collected by the smaller end of the tapered faceplate and is delivered to an opto-electronic sensor-array to be digitally sampled. Using a compressive sampling algorithm, we decode these raw lensfree images to validate the resolution (<4 μm) of this on-chip fluorescent imaging platform using microparticles as well as labeled Giardia muris cysts. This wide-field lensfree fluorescent microscopy platform, being compact and high-throughput, might provide a valuable tool especially for cytometry, rare cell analysis (involving large area microfluidic systems) as well as for microarray imaging applications. PMID:21283900

  12. Correlation between polarization sensitive optical coherence tomography and second harmonic generation microscopy in skin

    PubMed Central

    Le, Viet-Hoan; Lee, Seunghun; Kim, Bumju; Yoon, Yeoreum; Yoon, Calvin J.; Chung, Wan Kyun; Kim, Ki Hean

    2015-01-01

    Both polarization sensitive optical coherence tomography (PS-OCT) and second harmonic generation (SHG) microscopy are 3D optical imaging methods providing information related to collagen in the skin. PS-OCT provides birefringence information which is due to the collagen composition of the skin. SHG microscopy visualizes collagen fibers in the skin based on their SHG property. These two modalities have been applied to the same skin pathologies associated with collagen changes, but their relationship has not been examined. In this study, we tried to find the relationship by imaging the same skin samples with both modalities. Various parts of the normal rat skin and burn damaged skin were imaged ex vivo, and their images were analyzed both qualitatively and quantitatively. PS-OCT images were analyzed to obtain tissue birefringence. SHG images were analyzed to obtain collagen orientation indices by applying 2D Fourier transform. The skin samples having higher birefringence values had higher collagen orientation indices, and a linear correlation was found between them. Burn damaged skin showed decreases in both parameters compared to the control skins. This relationship between the bulk and microscopic properties of skin may be useful for further skin studies. PMID:26203380

  13. Potential Challenges in Near-Field Scanning Optical Microscopy for Space Applications

    NASA Technical Reports Server (NTRS)

    Vikram, C. S.; Witherow, W.

    2000-01-01

    Near-field scanning optical microscopy is a relatively new but very powerful technique for obtaining several metrological parameters at nanometer range spatial resolution. It is logical to think deploying it into space applications like diagnostics of protein crystal growth under microgravity conditions. One may attempt to deploy existing instrumentation and expect some results. However, the existing technology and commercial instrumentation is tailored to ground based laboratory situations. Even in those laboratory conditions, the role of fluids (common in crystal growth), rough objects (such as a crystal under growth), etc. on the instrumentation is only recently being investigated. These aspects combined with effects of reduced gravity environment are going to make the problem more complex. These technological challenges must be tackled for meaningful system operation in space. Since the microscopy concept has not been attempted so far in space, all of the actual problems are unknown. Nevertheless, based on current literature, some possible problems and potential solutions are described here. One may use the discussion for system modification/optimization during initial use of this kind of microscopy in space.

  14. Multiphoton microscopy, fluorescence lifetime imaging and optical spectroscopy for the diagnosis of neoplasia

    NASA Astrophysics Data System (ADS)

    Skala, Melissa Caroline

    2007-12-01

    Cancer morbidity and mortality is greatly reduced when the disease is diagnosed and treated early in its development. Tissue biopsies are the gold standard for cancer diagnosis, and an accurate diagnosis requires a biopsy from the malignant portion of an organ. Light, guided through a fiber optic probe, could be used to inspect regions of interest and provide real-time feedback to determine the optimal tissue site for biopsy. This approach could increase the diagnostic accuracy of current biopsy procedures. The studies in this thesis have characterized changes in tissue optical signals with carcinogenesis, increasing our understanding of the sensitivity of optical techniques for cancer detection. All in vivo studies were conducted on the dimethylbenz[alpha]anthracene treated hamster cheek pouch model of epithelial carcinogenesis. Multiphoton microscopy studies in the near infrared wavelength region quantified changes in tissue morphology and fluorescence with carcinogenesis in vivo. Statistically significant morphological changes with precancer included increased epithelial thickness, loss of stratification in the epithelium, and increased nuclear diameter. Fluorescence changes included a statistically significant decrease in the epithelial fluorescence intensity per voxel at 780 nm excitation, a decrease in the fluorescence lifetime of protein-bound nicotinamide adenine dinucleotide (NADH, an electron donor in oxidative phosphorylation), and an increase in the fluorescence lifetime of protein-bound flavin adenine dinucleotide (FAD, an electron acceptor in oxidative phosphorylation) with precancer. The redox ratio (fluorescence intensity of FAD/NADH, a measure of the cellular oxidation-reduction state) did not significantly change with precancer. Cell culture experiments (MCF10A cells) indicated that the decrease in protein-bound NADH with precancer could be due to increased levels of glycolysis. Point measurements of diffuse reflectance and fluorescence spectra in

  15. Optical and mechanical detection of near-field light by atomic force microscopy using a piezoelectric cantilever

    NASA Astrophysics Data System (ADS)

    Satoh, Nobuo; Kobayashi, Kei; Watanabe, Shunji; Fujii, Toru; Matsushige, Kazumi; Yamada, Hirofumi

    2016-08-01

    In this study, we developed an atomic force microscopy (AFM) system with scanning near-field optical microscopy (SNOM) using a microfabricated force-sensing cantilever with a lead zirconate titanate (PZT) thin film. Both optical and mechanical detection techniques were adopted in SNOM to detect scattered light induced by the interaction of the PZT cantilever tip apex and evanescent light, and SNOM images were obtained for each detection scheme. The mechanical detection technique did allow for a clear observation of the light scattered from the PZT cantilever without the interference observed by the optical detection technique, which used an objective lens, a pinhole, and a photomultiplier tube.

  16. A relation between cell cycle and intestinal metaplasia in oesophageal biopsies using optical and digital microscopy.

    PubMed

    Máté, Miklós; Molnár, Béla

    2015-07-01

    Protein expression changes in relation to cell cycles provide important information, and it may represent a new method for an early diagnosis of metaplasia - dysplasia - adenocarcinoma sequence. We investigated potential changes in cell cycle genes such as protooncogenes (PCNA, EGFR), tumour suppressor gene (p53), apoptotic TUNNEL (Tdt mediated dUTP nick and labelling) gene, as well as small intestinal mucus antigen (SIMA) and large intestinal mucus antigen (LIMA), which accumulates in metaplastic epithelium due to the inflammatory process in routine oesophageal biopsies using immunohistochemistry. Oesophageal biopsies were taken from patients with Barrett's oesophagus (n = 30), reflux oesophagitis (n = 30), healthy oesophagus (n = 30) and healthy cardia (n = 10). Immunohistochemical signalling was carried out by Streptavidin-Biotin-AEC (aminoetil-carbazol). Expression of PCNA was statistically significantly lower in healthy oesophagus (p < 0.05) versus reflux oesophagitis and Barrett's oesophagus. However, no significant change was detected in the expression of SIMA and LIMA in intestinal metaplasia. Further, EGFR, p53 and TUNNEL levels were significantly different in healthy versus Barrett's oesophagus. Manual counting using virtual microscopy was comparable with the result using conventional light microscopy, but the former is significantly quicker. There was no difference between manual and automated cell counting (p > 0.05). PMID:25740071

  17. Histone deacetylase expression patterns in developing murine optic nerve

    PubMed Central

    2014-01-01

    Background Histone deacetylases (HDACs) play important roles in glial cell development and in disease states within multiple regions of the central nervous system. However, little is known about HDAC expression or function within the optic nerve. As a first step in understanding the role of HDACs in optic nerve, this study examines the spatio-temporal expression patterns of methylated histone 3 (K9), acetylated histone 3 (K18), and HDACs 1–6 and 8–11 in the developing murine optic nerve head. Results Using RT-qPCR, western blot and immunofluorescence, three stages were analyzed: embryonic day 16 (E16), when astrocyte precursors are found in the optic stalk, postnatal day 5 (P5), when immature astrocytes and oligodendrocytes are found throughout the optic nerve, and P30, when optic nerve astrocytes and oligodendrocytes are mature. Acetylated and methylated histone H3 immunoreactivity was co-localized in the nuclei of most SOX2 positive glia within the optic nerve head and adjacent optic nerve at all developmental stages. HDACs 1–11 were expressed in the optic nerve glial cells at all three stages of optic nerve development in the mouse, but showed temporal differences in overall levels and subcellular localization. HDACs 1 and 2 were predominantly nuclear throughout optic nerve development and glial cell maturation. HDACs 3, 5, 6, 8, and 11 were predominantly cytoplasmic, but showed nuclear localization in at least one stage of optic nerve development. HDACs 4, 9 and10 were predominantly cytoplasmic, with little to no nuclear expression at any time during the developmental stages examined. Conclusions Our results showing that HDACs 1, 2, 3, 5, 6, 8, and 11 were each localized to the nuclei of SOX2 positive glia at some stages of optic nerve development and maturation and extend previous reports of HDAC expression in the aging optic nerve. These HDACs are candidates for further research to understand how chromatin remodeling through acetylation, deacetylation

  18. Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy.

    PubMed

    Huang, Chao; Sachse, Frank B; Hitchcock, Robert W; Kaza, Aditya K

    2016-01-01

    Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2 ± 0.3% and 98.0 ± 0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2 ± 0.3% and 94.0 ± 2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease. PMID:26808149

  19. Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy

    PubMed Central

    Huang, Chao; Sachse, Frank B.; Hitchcock, Robert W.; Kaza, Aditya K.

    2016-01-01

    Disturbances of the cardiac conduction system constitute a major risk after surgical repair of complex cases of congenital heart disease. Intraoperative identification of the conduction system may reduce the incidence of these disturbances. We previously developed an approach to identify cardiac tissue types using fiber-optics confocal microscopy and extracellular fluorophores. Here, we applied this approach to investigate sensitivity and specificity of human and automated classification in discriminating images of atrial working myocardium and specialized tissue of the conduction system. Two-dimensional image sequences from atrial working myocardium and nodal tissue of isolated perfused rodent hearts were acquired using a fiber-optics confocal microscope (Leica FCM1000). We compared two methods for local application of extracellular fluorophores: topical via pipette and with a dye carrier. Eight blinded examiners evaluated 162 randomly selected images of atrial working myocardium (n = 81) and nodal tissue (n = 81). In addition, we evaluated the images using automated classification. Blinded examiners achieved a sensitivity and specificity of 99.2±0.3% and 98.0±0.7%, respectively, with the dye carrier method of dye application. Sensitivity and specificity was similar for dye application via a pipette (99.2±0.3% and 94.0±2.4%, respectively). Sensitivity and specificity for automated methods of tissue discrimination were similarly high. Human and automated classification achieved high sensitivity and specificity in discriminating atrial working myocardium and nodal tissue. We suggest that our findings facilitate clinical translation of fiber-optics confocal microscopy as an intraoperative imaging modality to reduce the incidence of conduction disturbances during surgical correction of congenital heart disease. PMID:26808149

  20. Full-field illumination approach with multiple speckle for optical-resolution photoacoustic microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Poisson, Florian; Bossy, Emmanuel

    2016-03-01

    Optical-resolution photoacoustic endomicroscopy (OR-PAE) allows going beyond the limited penetration depth of conventional optical-resolution photoacoustic systems. Recently, it has been shown that OR-PAE may be performed through minimally invasive multimode fibers, by raster scanning a focus spot with optical wavefront shaping [1]. Here we introduce for the first time an approach to perform OR-PAE through a multimode fiber with a full-field illumination approach. By using multiple known speckle patterns, we show that it is possible to obtain optical-diffraction limited photoacoustic images, with the same resolution as that obtained by raster scanning a focus spot, i.e that of the speckle grain size. The fluctuations patterns of the photoacoustic amplitude at each pixel in the sample plane with the series of multiple speckle illumination were used to encode each pixel. This approach with known speckle illumination requires an initial calibration stage, that consists in learn a set of fluctuation patterns pixel per pixel, which will encode patterns each pixel of the scanned area. A point-like absorber was scanned across the filed-of-view during the calibration stage to acquire the reference patterns. Image reconstruction may be carried out by cross-correlating the series of photoacoustic amplitude measured with the sample to the reference patterns obtained during the calibration stage. In this work, the approach above was carried out both theoretically with Monte-carlo simulations and experimentally through a multi-mode fiber with samples made of absorbing spheres. [1] Papadopoulos et al., " Optical-resolution photoacoustic microscopy by use of a multimode fiber", Appl. Phys. Lett., 102(21), 2013

  1. Corneal imaging and refractive index measurement using a combined multiphoton microscopy and optical coherence tomography system

    NASA Astrophysics Data System (ADS)

    Lai, Tom; Chong, Shau Poh; Zhou, Yifeng; Moloney, Gregory; Tang, Shuo

    2013-02-01

    Refractive index (RI) is the optical property of a medium that describes its ability to bend incident light. The corneal refractive index is an especially important measurement in corneal and intraocular refractive surgery where its precise estimation is necessary to obtain accurate surgical outcomes. In this study, we calculated the corneal RI using a combined multiphoton microscopy (MPM) and optical coherence tomography (OCT) system. MPM excites and detects nonlinear signals including two photon excitation fluorescence (TPEF) and second harmonic generation (SHG). TPEF signals are observed from NADH in the cytoplasm, allowing MPM to image the cellular structures in the corneal epithelium and endothelium. SHG signals are observed from collagen, an abundant connective tissue found in the stroma. Optical coherence tomography (OCT) produces cross-sectional, structural images based on the interference fringes created by the reflected light from the sample and reference arms. Our system uses a single sub-10 fs Ti: sapphire laser source which is good for both MPM excitation and OCT resolution. The MPM and OCT images are coregistered when they are taken successively because their axial resolutions are similar and the system shares the laser source and the scanning unit. We can calculate the RI by measuring the optical thickness and the optical path length of the cornea from the MPM and OCT images respectively. We have imaged and calculated the RI of murine and piscine corneas. We were able to see the epithelial, stromal, and endothelial layers and compare their relative thicknesses and the organization of the stromal collagen lamellae. Our results showed that our system can provide both functional and structural information about the cornea and measure the RI of multi-layered tissues.

  2. Optical resolution photoacoustic microscopy using a Blu-ray DVD pickup head

    NASA Astrophysics Data System (ADS)

    Li, Meng-Lin; Wang, Po-Hsun

    2014-03-01

    Optical resolution photoacoustic microscopy (OR-PAM) has been shown as a promising tool for label-free microvascular and single-cell imaging in clinical and bioscientific applications. However, most OR-PAM systems are realized by using a bulky laser for photoacoustic excitation. The large volume and high price of the laser may restrain the popularity of OR-PAM. In this study, we attempt to develop a compact, portable, and low cost OR-PAM based on a consumer Blu-ray (405 nm) DVD pickup head for label-free micro-vascular imaging and red-blood-cell related blood examination. According to the high optical absorption of the hemoglobin at 405 nm, the proposed OR-PAM has potential to be an alternative for the conventional optical microscopy in the examinations of hematological morphology for blood routine. We showed that the Blu-ray DVD pickup head owns the required laser energy and focusing optics for OR-PAM. The firmware of a Blu-ray DVD drive was modified to allow its pickup head to generate nano-second laser pulses with a tunable pulse repetition rate of >30 kHz and a tunable pulse width ranging from 10 to 30 ns. The laser beam was focused onto the target after passing through a transparent cover slide, and then aligned to be confocal with a 50-MHz focused ultrasonic transducer in forward mode. To keep the target on focus, a scan involving auto-tracking procedure was performed. The measured maximum achievable lateral resolution was 1 μm which was mainly limited by the minimum step size of the used motorized stage. A blood smear was imaged without any staining. The red blood cells were well resolved and the biconcave structure could be clearly visualized. In addition, to verify the in vivo imaging capability of the proposed OR-PAM, the micro-vasculature of a mouse ear was imaged without any contrast agent. The results showed that it performed better than a 200x digital optical microscope in terms of image contrast and vascular morphology. In summaries, the proposed OR

  3. A transparent broadband ultrasonic detector based on an optical micro-ring resonator for photoacoustic microscopy

    PubMed Central

    Li, Hao; Dong, Biqin; Zhang, Zhen; Zhang, Hao F.; Sun, Cheng

    2014-01-01

    Photoacoustic microscopy (PAM) does not rely on contrast agent to image the optical absorption contrast in biological tissue. It is uniquely suited for measuring several tissue physiological parameters, such as hemoglobin oxygen saturation, that would otherwise remain challenging. Researchers are designing new clinical diagnostic tools and multimodal microscopic systems around PAM to fully unleash its potential. However, the sizeable and opaque piezoelectric ultrasonic detectors commonly used in PAM impose a serious constraint. Our solution is a coverslip-style optically transparent ultrasound detector based on a polymeric optical micro-ring resonator (MRR) with a total thickness of 250 μm. It enables highly-sensitive ultrasound detection over a wide receiving angle with a bandwidth of 140 MHz, which corresponds to a photoacoustic saturation limit of 287 cm−1, at an estimated noise-equivalent pressure (NEP) of 6.8 Pa. We also established a theoretical framework for designing and optimizing the MRR for PAM. PMID:24675547

  4. Sub-cellular quantitative optical diffraction tomography with digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Charrière, Florian; Kühn, Jonas; Colomb, Tristan; Cuche, Etienne; Marquet, Pierre; Depeursinge, Christian

    2007-02-01

    Digital holographic microscopy (DHM) is an interferometric technique, providing quantitative mapping of the phase shift induced by semi-transparent microscopic specimens, such as cells, with sub-wavelength resolution along the optical axis. Thanks to actual PCs and CCDs, DHM provides nowadays cost-effective instruments for real-time measurements at very high acquisition rates, with sub-micron transverse resolution. However, DHM phase images do not reveal the threedimensional (3D) internal distribution of refractive index, but a phase shift resulting from a mean refractive index (RI) integrated over the cellular thickness. Standard optical diffraction tomography (ODT) techniques can be efficiently applied to reveal internal structures and to measure 3D RI spatial distributions, by recording 2D DHM phase data for different sample orientations or illumination beam direction, in order to fill up entirely the Ewald sphere in the Fourier space. The 3D refractive index can then be reconstructed, even in the direct space with backpropagation algorithms or from the Fourier space with inverse Fourier transform. The presented technique opens wide perspectives in 3D cell imaging: the DHM-based micro-tomography furnishes invaluable data on the cell components optical properties, potentially leading to information about organelles intracellular distribution. Results obtained on biological specimens will be presented. Morphometric measurements can be extracted from the tomographic data, by detection based on the refractive index contrast within the 3D reconstructions. Results and perspectives about sub-cellular organelles identification inside the cell will also be exposed.

  5. Comparison of rotational imaging optical coherence tomography and selective plane illumination microscopy for embryonic study

    NASA Astrophysics Data System (ADS)

    Wu, Chen; Ran, Shihao; Le, Henry H.; Singh, Manmohan; Larina, Irina V.; Mayerich, David; Dickinson, Mary E.; Larin, Kirill V.

    2016-03-01

    The mouse is a common model for studying developmental diseases. Different optical techniques have been developed to investigate mouse embryos, but each has its own set of limitations and restrictions. In this study, we imaged the same E9.5 mouse embryo with rotational imaging Optical Coherence Tomography (RI-OCT) and Selective Plane Illumination Microscopy (SPIM), and compared the two techniques. Results demonstrate that both methods can provide images with micrometer-scale spatial resolution. The RI-OCT technique was developed to increase imaging depth of OCT by performing traditional OCT imaging at multiple sides and co-registering the images. In SPIM, optical sectioning is achieved by illuminating the sample with a sheet of light. In this study, the images acquired from both techniques are compared with each other to evaluate the benefits and drawbacks of each technique for embryonic imaging. Since 3D stacks can be obtained by SPIM from different angles by rotating the sample, it might be possible to build a hybrid setup of two imaging modalities to combine the advantages of each technique.

  6. Optically sectioned spatial-spectral coded holographic fluorescence microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Chen, Hsi-Hsun; Lin, Chen-Yen; Lin, Wei Tang; Luo, Yuan

    2016-03-01

    Wide-field fluorescent imaging severely suffers low resolution and poor contrast from out-of-focus background to image biological samples. In order to enhance optical sectioning capability, Confocal approach has been developed to filter out-of-focus background using point-to-point detection through a spatial pinhole. Recently, active structured illumination in wide-field fashion has been developed to reduce the transversal scanning cost, but still requires scanning in axial direction. Here, we present a wide-field multi-focal fluorescence microscopy incorporating spatial-spectral volume holographic gratings (MVHGs) with 3D active structured illumination to obtain optically sectioned images without scanning is presented. In contrast to conventional holographic techniques, which in general can not obtain fluorescence images, our approach does not require the formation of a hologram during imaging and is compatible with fluorescence based methods of imaging. Our approach requires pair-wise multi-depth resolved images, one with 3D active illumination, and the other with standard uniform illumination. Our approach is configured such that 3D illuminated planes occur inside the specimen, and also serve as the structured modulation for multiple axial planes imaged by MVHGs and display laterally onto the camera. The system can also be combined with micro-objective and relay systems for endoscopic operation. We demonstrate the proposed system's ability to simultaneously obtain wide-field, optically sectioned, and multi-depth resolved images of fluorescently labeled tissue structures.

  7. Keeping Track of the Selenide zoo. A Combined Optical Microscopy - EPMA Study of Complex Selenides

    NASA Astrophysics Data System (ADS)

    Schlothauer, T.; Renno, A. D.; Heide, G.

    2007-12-01

    Hunting for new mineral phases is a fascinating scientific activity. This kind of research not only serves the replenishment of mineralogy textbooks with new mineral names but also the industry with new potential semiconductors, laser crystals and other 'high-tech phases'. Chemical analyses using the electron microprobe are an essential intermediate step in the course of the description of a new mineral. The study of a great number of different Cu-Pb-Ag-As-Hg-Tl-Sb-Bi-Cd selenides from the former uranium deposit Schlema-Alberode (Saxony) in the German part of the Erzgebirge represented a twofold challenge to us. The complex genetic and age relations of the ore minerals and gangue minerals entailed the development of very complex microstructures in a tight space. Typical features are symplectitic intergrowths, exsolutions, fine lamellae, zoned crystals and the development of pseudo- and paramorphs. Altogether we found 34 different selenide, sulfide and arsenide minerals, including 6 dimorpheous phases. Many of these minerals are indistinguishable by electron-optical methods used during different stages of the study. Dimorpheous minerals like bellidoite and berzelianite are as much indistinguishable like intergrowths of the minerals berzelianite, mgriite and lollingite using backscattered and secondary electron images. Optical microscopy is the key to overcome these problems. We show that the step-by-step combination of polarized light microscopy, phase contrast microscopy and differential interference contrast microscopy using transmitted and reflected light allowed a secure discrimination of the different minerals and unknown phases. 16 elements are incorporated into these phases either as main, minor or trace elements. A multitude of overlapping peaks, potenzial fluorescence effects caused by adjacent phases and different matrices in the minerals demanded the development of several specific methods optimized for the analysis of Pb-Se-, Cu-Tl-Se-, Cu

  8. Understanding Alterations in Cell Nano-architecture during Early Carcinogenesis using Optical Microscopy

    NASA Astrophysics Data System (ADS)

    Damania, Dhwanil

    Carcinogenesis is a complex multi-step process which eventually results in a malignant phenotype that often progresses into a fatal metastatic stage. There are several molecular changes (e.g. DNA methylation, activation of proto-oncogenes, loss of tumor-suppressor genes, histone acetylation) that occur in cells prior to the microscopically detectable morphological alterations. Hence, it is intuitive that these molecular changes should impact various biochemical, biophysical and transport processes within the cell and therefore its nanoscale morphology. Furthermore, recent studies have established that apparently `normal' cells (i.e., away from the actual tumor location) undergo similar genetic/epigenetic changes as the actual cancer cells, giving rise to the phenomenon of field carcinogenesis. Unfortunately, traditional microscopy or histopathology cannot resolve structures below 300 nm due to diffraction-limited resolution. Hence, we developed a novel optical imaging technique, partial wave spectroscopic (PWS) microscopy or optical nanocytology which quantifies the nanoscale refractive-index fluctuations (i.e. mass-density variations such as chromatin compaction) in an optically measured biomarker, disorder strength (Ld). This dissertation proves the nanoscale sensitivity of PWS nanocytology and shows that increase in Ld parallels neoplastic potential of a cell by using standardized cell-lines and animal-models. Based on concept of field carcinogenesis, we employ PWS nanocytology in a multi-center clinical study on approximately 450 patients in four different cancer-types (colon, ovarian, thyroid and lung) and we illustrate that nanoscale disorder increase is a ubiquitous phenomenon across different organs. We further demonstrate the potential of PWS nanocytology in predicting risk for developing future neoplasia. Biologically, we prove that cytoskeletal organization in both nucleus and cytoplasm plays a crucial role in governing L d-differences. Moreover, we

  9. Simulation of near-field scanning optical microscopy using a plasmonic gap probe

    NASA Astrophysics Data System (ADS)

    Tanaka, Kazuo; Tanaka, Masahiro; Katayama, Kiyofumi

    2006-10-01

    Imaging by near-field scanning optical microscopy (NSOM) with a plasmonic gap probe (PGP) is simulated to confirm the operation of the recently proposed PGP. The simulations demonstrate that the probe works in illumination, collection-reflection and collection mode, and that is it not necessary to vibrate the probe tip in order to remove background noise. The resolution of the scanned image is also shown to be approximately equal to the diameter of the probe tip. Furthermore, the throughput of the probe is much higher than conventional aperture probes providing similar resolution. The proposed probe thus has the advantages of both aperture probes and scattering probes, and is expected to have excellent characteristics for use as a scanning probe for NSOM.

  10. A paper microfluidic cartridge for automated staining of malaria parasites with an optically transparent microscopy window.

    PubMed

    Horning, Matthew P; Delahunt, Charles B; Singh, S Ryan; Garing, Spencer H; Nichols, Kevin P

    2014-06-21

    A paper microfluidic cartridge for the automated staining of malaria parasites (Plasmodium) with acridine orange prior to microscopy is presented. The cartridge enables simultaneous, sub-minute generation of both thin and thick smears of acridine orange stained parasites. Parasites are stained in a cellulose matrix, after which the parasites are ejected via capillary forces into an optically transparent chamber. The unique slanted design of the chamber ensures that a high percentage of the stained blood will be of the required thickness for a thin smear, without resorting to spacers or other methods that can increase production cost or require tight quality controls. A hydrophobic snorkel facilitates the removal of air bubbles during filling. The cartridge contains both a thin smear region, where a single layer of cells is presented unobstructed, for ease of species identification, and a thick smear region, containing multiple cell layers, for enhanced limit of detection. PMID:24781199

  11. Portable optical-resolution photoacoustic microscopy with a pulsed laser diode excitation

    NASA Astrophysics Data System (ADS)

    Zeng, Lvming; Liu, Guodong; Yang, Diwu; Ji, Xuanrong

    2013-02-01

    Optical-resolution photoacoustic microscopy (OR-PAM) has been significantly improved in terms of spatial resolution, detection sensitivity, imaging speed, and penetration depth. However, the popular producibility of OR-PAM system is still limited by the size and cost of solid-state laser excitation. Here, we developed a portable laser-diode-based OR-PAM (LD-OR-PAM) system using a pulsed semiconductor laser source, which was operated at 905 ± 15 nm with a pulse energy as low as 4.9 μJ. The measured lateral resolution has been improved to ˜1.5 μm from hundreds of microns. The compact and inexpensive natures of LD-OR-PAM would promote the potential clinical applications such as in dermatology.

  12. Noncontact broadband all-optical photoacoustic microscopy based on a low-coherence interferometer

    NASA Astrophysics Data System (ADS)

    Chen, Zhongjiang; Yang, Sihua; Wang, Yi; Xing, Da

    2015-01-01

    We developed and fabricated a noncontact broadband all-optical photoacoustic microscopy (BD-AO-PAM) with a microchip laser and an all-fiber low coherence interferometer. Currently, the available detection bandwidth of the BD-AO-PAM is 67 MHz, and the lateral resolution measured by carbon fibers reaches 11 μm. Furthermore, the imaging capability of the BD-AO-PAM was testified by imaging hairs embedded in scattering gel and in vivo blood vessels of a mouse ear. The experimental results demonstrate that the BD-AO-PAM can image the tissues with high spatial resolution in vivo, which can be used as portable noncontact PAM for biomedical applications.

  13. Surface-polariton propagation for scanning near-field optical microscopy application.

    PubMed

    Keilmann, F

    1999-01-01

    Surface plasmon-, phonon- and exciton-polaritons exist on specific materials in specific spectral regions. We assess the properties of such travelling surface-bound electromagnetic waves relevant for scanning near-field optical microscopy applications, i.e. the tightness of surface binding, the attenuation, the phase velocity and the coupling with free-space electromagnetic waves. These quantities can be directly determined by photographic imaging of surface plasmon- and surface phonon-polaritons, in both the visible and mid-infared regions. Focusing of mid-infrared surface plasmons is demonstrated. Surface waveguides to transport and focus photons to the tip of a scanning near-field probe are outlined. PMID:11388308

  14. High-resolution full-field optical coherence microscopy using a broadband light-emitting diode.

    PubMed

    Ogien, Jonas; Dubois, Arnaud

    2016-05-01

    High-resolution full-field optical coherence microscopy (FF-OCM) is demonstrated using a single broadband light-emitting diode (LED). The characteristics of the LED-illumination FF-OCM system are measured and compared to those obtained using a halogen lamp, the light source of reference in FF-OCM. Both light sources yield identical performance in terms of spatial resolution and detection sensitivity, using the same setup and camera. In particular, an axial resolution of 0.7 μm (in water) is reached. A Xenopus laevis tadpole and ex-vivo human skin have been imaged using both sources, resulting in similar images, showing for the first time that LEDs could favorably replace halogen lamps in high-resolution FF-OCM for biomedical imaging. PMID:27137603

  15. Effect of clay on structure of epoxy/PCL nanocomposite: In situ optical microscopy study

    NASA Astrophysics Data System (ADS)

    Rotrekl, Jakub; Kelnar, Ivan

    2012-07-01

    The effect of clay on reaction induced phase separation (RIPS) mechanism and resulting morphology of diglycidyl ether of bisphenol-A based epoxy containing poly(ɛ-caprolactone)(PCL) was investigated by optical microscopy. At PCL concentration lower than 10%, the presence of the clay leads to refinement of PCL inclusions, while at 15% and 20% the clay causes the phase inversion of structure consisting of PCL rich matrix with rough epoxy globules to the fine PCL inclusions in epoxy matrix. At 30% PCL content, the influence of clay is analogical but leads to the transformation into co-continous structure only. These changes in morphology seem to be a consequence of the change in dynamic asymmetry of the system, caused by affecting of the epoxy parameters by clay.

  16. Adaptive optics two photon microscopy with direct wavefront sensing using autofluorescent guide-stars

    NASA Astrophysics Data System (ADS)

    Tao, Xiaodong; Norton, Andrew; Kissel, Matthew; Azucena, Oscar; Kubby, Joel

    2014-03-01

    A fast direct wavefront sensing method for dynamic in-vivo adaptive optical two photon microscopy has demonstrated. By using the direct wavefront sensing and open loop control, the system provides high-speed wavefront measurement and correction. To measure the wavefront in the middle of a Drosophila embryo at early stages, autofluorescence from endogenous fluorophores in the yolk were used as reference guide-stars. This method does not rely on fluorescently labeled proteins as guide-stars, which can simplify the sample preparation for wavefront measurement. The method was tested through live imaging of a Drosophila embryo. The aberration in the middle of the embryo was measured directly for the first time. After correction, both contrast and signal intensity of the structure in the middle of the embryo was improved.

  17. Label-free imaging of developing vasculature in zebrafish with phase variance optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Yu; Fingler, Jeff; Trinh, Le A.; Fraser, Scott E.

    2016-03-01

    A phase variance optical coherence microscope (pvOCM) has been created to visualize blood flow in the vasculature of zebrafish embryos, without using exogenous labels. The pvOCM imaging system has axial and lateral resolutions of 2 μm in tissue, and imaging depth of more than 100 μm. Imaging of 2-5 days post-fertilization zebrafish embryos identified the detailed structures of somites, spinal cord, gut and notochord based on intensity contrast. Visualization of the blood flow in the aorta, veins and intersegmental vessels was achieved with phase variance contrast. The pvOCM vasculature images were confirmed with corresponding fluorescence microscopy of a zebrafish transgene that labels the vasculature with green fluorescent protein. The pvOCM images also revealed functional information of the blood flow activities that is crucial for the study of vascular development.

  18. Optical third-harmonic surface microscopy using ultra-short laser pulses

    NASA Astrophysics Data System (ADS)

    Stoker, David; Downer, Michael C.; Becker, Michael F.; Keto, John W.

    2005-12-01

    The time dependence of third-harmonic generation in an optical material is modelled for an ultrafast pulse from a Ti : Sapphire laser. For normally dispersive materials, the group velocity mismatch of the fundamental and harmonic fields is significant. In the absence of phase matching, pulse breakup is observed. Thirdharmonic generation is governed by a bulk susceptibility, but is sensitive to material interfaces. It is shown that the sensitivity to interfaces is enhanced by group velocity walk-off, and that interface resolution is better than previously reported. It is possible to use the surface sensitivity to develop an interferometric microscopy tool for investigating thin films. The experimental technique is demonstrated on a thin AlN coating grown on a Al2O3 substrate.

  19. Towards controlling molecular motions in fluorescence microscopy and optical trapping: a spatiotemporal approach

    PubMed Central

    Kumar De, Arijit; Goswami, Debabrata

    2013-01-01

    This account reviews some recent studies pursued in our group on several control experiments with important applications in (one-photon) confocal and two-photon fluorescence laser-scanning microscopy and optical trapping with laser tweezers. We explore the simultaneous control of internal and external (i.e. centre-of-mass motion) degrees of freedom, which require the coupling of various control parameters to result in the spatiotemporal control. Of particular interest to us is the implementation of such control schemes in living systems. A live cell is a system of a large number of different molecules which combine and interact to generate complex structures and functions. These combinations and interactions of molecules need to be choreographed perfectly in time and space to achieve intended intra-cellular functions. Spatiotemporal control promises to be a versatile tool for dynamical control of spatially manipulated bio-molecules. PMID:23814326

  20. Primary ciliary dyskinesia assessment by means of optical flow analysis of phase-contrast microscopy images.

    PubMed

    Parrilla, Eduardo; Armengot, Miguel; Mata, Manuel; Sánchez-Vílchez, José Manuel; Cortijo, Julio; Hueso, José L; Riera, Jaime; Moratal, David

    2014-04-01

    Primary ciliary dyskinesia implies cilia with defective or total absence of motility, which may result in sinusitis, chronic bronchitis, bronchiectasis and male infertility. Diagnosis can be difficult and is based on an abnormal ciliary beat frequency (CBF) and beat pattern. In this paper, we present a method to determine CBF of isolated cells through the analysis of phase-contrast microscopy images, estimating cilia motion by means of an optical flow algorithm. After having analyzed 28 image sequences (14 with a normal beat pattern and 14 with a dyskinetic pattern), the normal group presented a CBF of 5.2 ± 1.6 Hz, while the dyskinetic patients presented a 1.9 ± 0.9 Hz CBF. The cutoff value to classify a dyskinetic specimen was set to 3.45 Hz (sensitivity 0.86, specificity 0.93). The presented methodology has provided excellent results to objectively diagnose PCD. PMID:24438822

  1. Collagen remodeling in photo-thermal damaged skin with optical coherence tomography and multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Wu, Shu-lian; Li, Hui; Zhang, Xiao-man; Yu, Lili

    2009-08-01

    Cutaneous photo-thermal damage is the common damages in clinical medicine; it is a complex and dynamic process that follows an orderly sequence of events. The sequence can be roughly divided into three distinct, yet sequentially overlapping phases-inflammation, granulation tissue formation, and tissue remodeling. Characteristic structural changes associated with each phase could provide a basis for photo-thermal damage assessment with imaging technologies. Monitoring the skin tissue response during the skin after irradiated by laser and tracing the process of skin remodeling would help to understand the mechanism of photo-thermal. Optical coherence tomography (OCT) and multiphoton microscopy (MPM) imaging were used to observe the process of the collagen remodeling in mouse dermis photo-thermal injured which after irradiated by intense pulsed light source (IPLs) in this paper. Our finding showed that the OCT and MPM techniques can image the process of collagen remodeling in mouse dermis.

  2. Simultaneous optical coherence and multiphoton microscopy of skin-equivalent tissue models

    NASA Astrophysics Data System (ADS)

    Barton, Jennifer K.; Tang, Shuo; Lim, Ryan; Tromberg, Bruce J.

    2007-07-01

    Three-layer skin-equivalent models (rafts) were created consisting of a collagen/fibroblast layer and an air-exposed keratinocyte layer. Rafts were imaged with a tri-modality microscope including optical coherence (OC), two-photon excited fluorescence (TPEF), and second harmonic generation (SHG) channels. Some rafts were stained with Hoechst 33343 or rhodamine 123, and some were exposed to dimethyl sulfoxide (DMSO). OC microscopy revealed signal in cell cytoplasm and nuclear membranes, and a characteristic texture in the collagen/fibroblast layer. TPEF showed signal in cell cytoplasm and from collagen, and stained specimens revealed cell nuclei or mitochondria. There was little SHG in the keratinocyte layer, but strong signal from collagen bundles. Endogenous signals were severely attenuated in DMSO treated rafts; stained samples revealed shrunken and distorted cell structure. OC, TPEF, and SHG can provide complementary and non-destructive information about raft structure and effect of chemical agents.

  3. Macro-optical trapping for sample confinement in light sheet microscopy

    PubMed Central

    Yang, Zhengyi; Piksarv, Peeter; Ferrier, David E.K.; Gunn-Moore, Frank J.; Dholakia, Kishan

    2015-01-01

    Light sheet microscopy is a powerful approach to construct three-dimensional images of large specimens with minimal photo-damage and photo-bleaching. To date, the specimens are usually mounted in agents such as agarose, potentially restricting the development of live samples, and also highly mobile specimens need to be anaesthetized before imaging. To overcome these problems, here we demonstrate an integrated light sheet microscope which solely uses optical forces to trap and hold the sample using a counter-propagating laser beam geometry. Specifically, tobacco plant cells and living Spirobranchus lamarcki larvae were successfully trapped and sectional images acquired. This novel approach has the potential to significantly expand the range of applications for light sheet imaging. PMID:26309743

  4. Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart

    PubMed Central

    Huang, Chao; Kaza, Aditya K.; Hitchcock, Robert W.; Sachse, Frank B.

    2014-01-01

    Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5–9 lines, which is comparable to 4–8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery. PMID:25309455

  5. Elementary steps at the surface of ice crystals visualized by advanced optical microscopy

    PubMed Central

    Sazaki, Gen; Zepeda, Salvador; Nakatsubo, Shunichi; Yokoyama, Etsuro; Furukawa, Yoshinori

    2010-01-01

    Due to the abundance of ice on earth, the phase transition of ice plays crucially important roles in various phenomena in nature. Hence, the molecular-level understanding of ice crystal surfaces holds the key to unlocking the secrets of a number of fields. In this study we demonstrate, by laser confocal microscopy combined with differential interference contrast microscopy, that elementary steps (the growing ends of ubiquitous molecular layers with the minimum height) of ice crystals and their dynamic behavior can be visualized directly at air-ice interfaces. We observed the appearance and lateral growth of two-dimensional islands on ice crystal surfaces. When the steps of neighboring two-dimensional islands coalesced, the contrast of the steps always disappeared completely. We were able to discount the occurrence of steps too small to detect directly because we never observed the associated phenomena that would indicate their presence. In addition, classical two-dimensional nucleation theory does not support the appearance of multilayered two-dimensional islands. Hence, we concluded that two-dimensional islands with elementary height (0.37 and 0.39 nm on basal and prism faces, respectively) were visualized by our optical microscopy. On basal and prism faces, we also observed the spiral growth steps generated by screw dislocations. The distance between adjacent spiral steps on a prism face was about 1/20 of that on a basal face. Hence, the step ledge energy of a prism face was 1/20 of that on a basal face, in accord with the known lower-temperature roughening transition of the prism face. PMID:20974928

  6. Correlative nonlinear optical microscopy and infrared nanoscopy reveals collagen degradation in altered parchments

    NASA Astrophysics Data System (ADS)

    Latour, Gaël; Robinet, Laurianne; Dazzi, Alexandre; Portier, François; Deniset-Besseau, Ariane; Schanne-Klein, Marie-Claire

    2016-05-01

    This paper presents the correlative imaging of collagen denaturation by nonlinear optical microscopy (NLO) and nanoscale infrared (IR) spectroscopy to obtain morphological and chemical information at different length scales. Such multiscale correlated measurements are applied to the investigation of ancient parchments, which are mainly composed of dermal fibrillar collagen. The main issue is to characterize gelatinization, the ultimate and irreversible alteration corresponding to collagen denaturation to gelatin, which may also occur in biological tissues. Key information about collagen and gelatin signatures is obtained in parchments and assessed by characterizing the denaturation of pure collagen reference samples. A new absorbing band is observed near the amide I band in the IR spectra, correlated to the onset of fluorescence signals in NLO images. Meanwhile, a strong decrease is observed in Second Harmonic signals, which are a structural probe of the fibrillar organization of the collagen at the micrometer scale. NLO microscopy therefore appears as a powerful tool to reveal collagen degradation in a non-invasive way. It should provide a relevant method to assess or monitor the condition of collagen-based materials in museum and archival collections and opens avenues for a broad range of applications regarding this widespread biological material.

  7. Three-dimensional reconstructions from optical sections of thick mouse inner ears using confocal microscopy.

    PubMed

    Kopecky, B J; Duncan, J S; Elliott, K L; Fritzsch, B

    2012-12-01

    Three-dimensional (3D) reconstructions of the vertebrate inner ear have provided novel insights into the development of this complex organ. 3D reconstructions enable superior analysis of phenotypic differences between wild type and mutant ears but can result in laborious work when reconstructed from physically sectioned material. Although nondestructive optical sectioning light sheet microscopy may ultimately prove the ideal solution, these technologies are not yet commercially available, or in many instances are not monetarily feasible. Here we introduce a simple technique to image a fluorescently labelled ear at different stages throughout development at high resolution enabling 3D reconstruction of any component of the inner ear using confocal microscopy. We provide a step-by-step manual from tissue preparation to imaging to 3D reconstruction and analysis including a rationale and troubleshooting guide at each step for researchers with different equipment, protocols, and access to resources to successfully incorporate the principles of this method and customize them to their laboratory settings. PMID:23140378

  8. Accurate phase measurements for thick spherical objects using optical quadrature microscopy

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

    In vitro fertilization (IVF) procedures have resulted in the birth of over three million babies since 1978. Yet the live birth rate in the United States was only 34% in 2005, with 32% of the successful pregnancies resulting in multiple births. These multiple pregnancies were directly attributed to the transfer of multiple embryos to increase the probability that a single, healthy embryo was included. Current viability markers used for IVF, such as the cell number, symmetry, size, and fragmentation, are analyzed qualitatively with differential interference contrast (DIC) microscopy. However, this method is not ideal for quantitative measures beyond the 8-cell stage of development because the cells overlap and obstruct the view within and below the cluster of cells. We have developed the phase-subtraction cell-counting method that uses the combination of DIC and optical quadrature microscopy (OQM) to count the number of cells accurately in live mouse embryos beyond the 8-cell stage. We have also created a preliminary analysis to measure the cell symmetry, size, and fragmentation quantitatively by analyzing the relative dry mass from the OQM image in conjunction with the phase-subtraction count. In this paper, we will discuss the characterization of OQM with respect to measuring the phase accurately for spherical samples that are much larger than the depth of field. Once fully characterized and verified with human embryos, this methodology could provide the means for a more accurate method to score embryo viability.

  9. Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

    PubMed Central

    Matysik, Artur; Kraut, Rachel S.

    2014-01-01

    Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies. PMID:24961277

  10. Correlative nonlinear optical microscopy and infrared nanoscopy reveals collagen degradation in altered parchments.

    PubMed

    Latour, Gaël; Robinet, Laurianne; Dazzi, Alexandre; Portier, François; Deniset-Besseau, Ariane; Schanne-Klein, Marie-Claire

    2016-01-01

    This paper presents the correlative imaging of collagen denaturation by nonlinear optical microscopy (NLO) and nanoscale infrared (IR) spectroscopy to obtain morphological and chemical information at different length scales. Such multiscale correlated measurements are applied to the investigation of ancient parchments, which are mainly composed of dermal fibrillar collagen. The main issue is to characterize gelatinization, the ultimate and irreversible alteration corresponding to collagen denaturation to gelatin, which may also occur in biological tissues. Key information about collagen and gelatin signatures is obtained in parchments and assessed by characterizing the denaturation of pure collagen reference samples. A new absorbing band is observed near the amide I band in the IR spectra, correlated to the onset of fluorescence signals in NLO images. Meanwhile, a strong decrease is observed in Second Harmonic signals, which are a structural probe of the fibrillar organization of the collagen at the micrometer scale. NLO microscopy therefore appears as a powerful tool to reveal collagen degradation in a non-invasive way. It should provide a relevant method to assess or monitor the condition of collagen-based materials in museum and archival collections and opens avenues for a broad range of applications regarding this widespread biological material. PMID:27194180

  11. Correlative nonlinear optical microscopy and infrared nanoscopy reveals collagen degradation in altered parchments

    PubMed Central

    Latour, Gaël; Robinet, Laurianne; Dazzi, Alexandre; Portier, François; Deniset-Besseau, Ariane; Schanne-Klein, Marie-Claire

    2016-01-01

    This paper presents the correlative imaging of collagen denaturation by nonlinear optical microscopy (NLO) and nanoscale infrared (IR) spectroscopy to obtain morphological and chemical information at different length scales. Such multiscale correlated measurements are applied to the investigation of ancient parchments, which are mainly composed of dermal fibrillar collagen. The main issue is to characterize gelatinization, the ultimate and irreversible alteration corresponding to collagen denaturation to gelatin, which may also occur in biological tissues. Key information about collagen and gelatin signatures is obtained in parchments and assessed by characterizing the denaturation of pure collagen reference samples. A new absorbing band is observed near the amide I band in the IR spectra, correlated to the onset of fluorescence signals in NLO images. Meanwhile, a strong decrease is observed in Second Harmonic signals, which are a structural probe of the fibrillar organization of the collagen at the micrometer scale. NLO microscopy therefore appears as a powerful tool to reveal collagen degradation in a non-invasive way. It should provide a relevant method to assess or monitor the condition of collagen-based materials in museum and archival collections and opens avenues for a broad range of applications regarding this widespread biological material. PMID:27194180

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

    PubMed Central

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

    1994-01-01

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

  13. Nonlinear optical microscopy and microspectroscopy of oral precancers and early cancer

    NASA Astrophysics Data System (ADS)

    Vargas, Gracie; Edward, Kert

    2013-02-01

    Multiphoton autofluorescence microscopy (MPAM) offers the ability to assess morphometry similar to that of pathologic evaluation as well as biochemical information from endogenous fluorophores which are altered with neoplastic transformation. In this study the spectroscopic properties of normal and neoplastic oral epithelium were evaluated toward the goal of identifying image/spectroscopic based indicators of neoplastic transformation using nonlinear optical microscopy. Results indicated measureable differences between normal, dysplasia, and SCC that could be helpful in delineating between the three conditions. In particular, a blue shift in autofluorescence emission was experienced for dysplasia relative to normal. However, in the case of SCC the epithelial emission experienced a significant red shift relative to both dysplasia and normal and displayed in an additional red peak that was not present in either normal or dysplastic mucosa. Results were consistent with published results for SCC in the single-photon literature. The study demonstrates that multiphoton autofluorescence spectroscopy may reveal features of oral mucosa that can be useful for differentiating normal and neoplastic mucosa. When combined with morphometry provided by MPAM, a potentially powerful technique for imaging of the oral cavity could be developed which provides both morphometric and spectroscopic information.

  14. Measurement of intracellular calcium gradients in single living cells using optical sectioning microscopy

    NASA Astrophysics Data System (ADS)

    Yelamarty, Rao V.; Cheung, Joseph Y.

    1992-06-01

    Intracellular free calcium has been recognized as a regulator of many cellular processes and plays a key role in mediating actions of many drugs. To elucidate subcellular spatial calcium changes throughout the cell in three dimensions (3-D), optical sectioning microscopy was applied using digital imaging coupled fluorescence microscopy. The cell was loaded with a fluorescent indicator, fura-2, and a stack of sectional fluorescent images were acquired, digitized and finally stored on-line for post image analysis. Each sectional image was then deconvolved, to remove contaminating light signals from adjacent planes, using the Nearest Neighboring Deconvolution Algorithm (NNDA) and the overall imaging system's empirical Point Spread Function (PSF) that is measured with a 0.25 micrometers fluorescent bead. Using this technique, we measured that the addition of growth factors caused a 2 - 3 fold increase (1) in nuclear calcium compared to cytosolic calcium in blood cells and (2) in both nuclear and cytosolic calcium in liver cells. Such spatial information, which is important in understanding subcellular processes, would not be possible to measure with other methods.

  15. Three-dimensional simultaneous optical coherence tomography and confocal fluorescence microscopy for investigation of lung tissue

    NASA Astrophysics Data System (ADS)

    Gaertner, Maria; Cimalla, Peter; Meissner, Sven; Kuebler, Wolfgang M.; Koch, Edmund

    2012-07-01

    Although several strategies exist for a minimal-invasive treatment of patients with lung failure, the mortality rate of acute respiratory distress syndrome still reaches 30% at minimum. This striking number indicates the necessity of understanding lung dynamics on an alveolar level. To investigate the dynamical behavior on a microscale, we used three-dimensional geometrical and functional imaging to observe tissue parameters including alveolar size and length of embedded elastic fibers during ventilation. We established a combined optical coherence tomography (OCT) and confocal fluorescence microscopy system that is able to monitor the distension of alveolar tissue and elastin fibers simultaneously within three dimensions. The OCT system can laterally resolve a 4.9 μm line pair feature and has an approximately 11 μm full-width-half-maximum axial resolution in air. confocal fluorescence microscopy visualizes molecular properties of the tissue with a resolution of 0.75 μm (laterally), and 5.9 μm (axially) via fluorescence detection of the dye sulforhodamine B specifically binding to elastin. For system evaluation, we used a mouse model in situ to perform lung distension by application of different constant pressure values within the physiological regime. Our method enables the investigation of alveolar dynamics by helping to reveal basic processes emerging during artificial ventilation and breathing.

  16. Quantification of molecular diffusion in arterial tissues with optical coherence tomography and fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Ghosn, M. G.; Syed, S. H.; Befrui, N. A.; Leba, M.; Vijayananda, A.; Sudheendran, N.; Larin, K. V.

    2009-06-01

    Alternations in vascular permeability for different molecules, drugs, and contrast agents might be a significant early marker of development of various diseases such as atherosclerosis. However, up to date experimental studies of molecular diffusion across vascular wall have been limited. Recently, we demonstrated that the Optical Coherence Tomography (OCT) technique could be applied for noninvasive and nondestructive quantification of molecular diffusion in different biological tissues. However, the viability of the OCT-based assessment of molecular diffusion should be validated with established methods. This study focused on comparing molecular diffusion rates in vascular tissues measured with OCT and standard fluorescent microscopy. Noninvasive quantification of tetramethylrhodamine (fluorescent dye) permeability in porcine vascular tissues was performed using a fiber-based OCT system. Concurrently, standard histological examination of dye diffusion was performed and quantified with fluorescent microscopy. The permeability of tetramethylrhodamine was found to be (2.08 ± 0.31) × 10-5 cm/s with the fluorescent technique ( n = 8), and (2.45 ± 0.46) × 10-5 cm/s with the OCT ( n = 3). Good correlation between permeability rates measured by OCT and histology was demonstrated, suggesting that the OCT-based method could be used for accurate, nondestructive assessment of molecular diffusion in multilayered tissues.

  17. Programmable Colored Illumination Microscopy (PCIM): A practical and flexible optical staining approach for microscopic contrast enhancement

    NASA Astrophysics Data System (ADS)

    Zuo, Chao; Sun, Jiasong; Feng, Shijie; Hu, Yan; Chen, Qian

    2016-03-01

    Programmable colored illumination microscopy (PCIM) has been proposed as a flexible optical staining technique for microscopic contrast enhancement. In this method, we replace the condenser diaphragm of a conventional microscope with a programmable thin film transistor-liquid crystal display (TFT-LCD). By displaying different patterns on the LCD, numerous established imaging modalities can be realized, such as bright field, dark field, phase contrast, oblique illumination, and Rheinberg illuminations, which conventionally rely on intricate alterations in the respective microscope setups. Furthermore, the ease of modulating both the color and the intensity distribution at the aperture of the condenser opens the possibility to combine multiple microscopic techniques, or even realize completely new methods for optical color contrast staining, such as iridescent dark-field and iridescent phase-contrast imaging. The versatility and effectiveness of PCIM is demonstrated by imaging of several transparent colorless specimens, such as unstained lung cancer cells, diatom, textile fibers, and a cryosection of mouse kidney. Finally, the potentialities of PCIM for RGB-splitting imaging with stained samples are also explored by imaging stained red blood cells and a histological section.

  18. Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices

    SciTech Connect

    Lee, P.-F.; Yeh, Alvin T.; Bayless, Kayla J.

    2009-02-01

    The interactions between endothelial cells (ECs) and the extracellular matrix (ECM) are fundamental in mediating various steps of angiogenesis, including cell adhesion, migration and sprout formation. Here, we used a noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique to optically image endothelial sprouting morphogenesis in three-dimensional (3D) collagen matrices. We simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Dynamic 3D imaging revealed EC interactions with collagen fibers along with quantifiable alterations in collagen matrix density elicited by EC movement through and morphogenesis within the matrix. Specifically, we observed increased collagen density in the area between bifurcation points of sprouting structures and anisotropic increases in collagen density around the perimeter of lumenal structures, but not advancing sprout tips. Proteinase inhibition studies revealed membrane-associated matrix metalloproteinase were utilized for sprout advancement and lumen expansion. Rho-associated kinase (p160ROCK) inhibition demonstrated that the generation of cell tension increased collagen matrix alterations. This study followed sprouting ECs within a 3D matrix and revealed that the advancing structures recognize and significantly alter their extracellular environment at the periphery of lumens as they progress.

  19. In situ 3D characterization of historical coatings and wood using multimodal nonlinear optical microscopy.

    PubMed

    Latour, Gaël; Echard, Jean-Philippe; Didier, Marie; Schanne-Klein, Marie-Claire

    2012-10-22

    We demonstrate multimodal nonlinear optical imaging of historical artifacts by combining Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (2PEF) microscopies. We first identify the nonlinear optical response of materials commonly encountered in coatings of cultural heritage artifacts by analyzing one- and multi-layered model samples. We observe 2PEF signals from cochineal lake and sandarac and show that pigments and varnish films can be discriminated by exploiting their different emission spectral ranges as in luminescence linear spectroscopy. We then demonstrate SHG imaging of a filler, plaster, composed of bassanite particles which exhibit a non centrosymmetric crystal structure. We also show that SHG/2PEF imaging enables the visualization of wood microstructure through typically 60 µm-thick coatings by revealing crystalline cellulose (SHG signal) and lignin (2PEF signal) in the wood cell walls. Finally, in situ multimodal nonlinear imaging is demonstrated in a historical violin. SHG/2PEF imaging thus appears as a promising non-destructive and contactless tool for in situ 3D investigation of historical coatings and more generally for wood characterization and coating analysis at micrometer scale. PMID:23187225

  20. Acute changes associated with electrode insertion measured with optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Hammer, Daniel X.; Lozzi, Andrea; Boretsky, Adam; Agrawal, Anant; Welle, Cristin G.

    2016-03-01

    Despite advances in functional neural imaging, penetrating microelectrodes provide the most direct interface for the extraction of neural signals from the nervous system and are a critical component of many high degree-of-freedom braincomputer interface devices. Electrode insertion is a traumatic event that elicits a complex neuroinflammatory response. In this investigation we applied optical coherence microscopy (OCM), particularly optical coherence angiography (OCA), to characterize the immediate tissue response during microelectrode insertion. Microelectrodes of varying dimension and footprint (one-, two-, and four-shank) were inserted into mouse motor cortex beneath a window after craniotomy surgery. The microelectrodes were inserted in 3-4 steps at 15-20°, with approximately 250 μm linear insertion distance for each step. Before insertion and between each step, OCM datasets were collected, including for quantitative capillary velocimetry. A cohort of control animals without microelectrode insertion was also imaged over a similar time period (2-3 hours). Mechanical tissue deformation was observed in all the experimental animals. The quantitative angiography results varied across animals, and were not correlated with device dimensions. In some cases, localized flow drop-out was observed in a small region surrounding the electrode, while in other instances a global disruption in flow occurred, perhaps as a result of large vessel compression caused by mechanical pressure. OCM is a tool that can be used in various neurophotonics applications, including quantification of the neuroinflammatory response to penetrating electrode insertion.

  1. In vivo optical resolution photoacoustic microscopy using glancing angle-deposited nanostructured Fabry-Perot etalons.

    PubMed

    Hajireza, Parsin; Sorge, Jason; Brett, Michael; Zemp, Roger

    2015-04-01

    In this Letter, reflection-mode optical resolution photoacoustic microscopy (OR-PAM) using glancing angle-deposited (GLAD) nanostructured Fabry-Perot interferometers (FPI) for in vivo applications is reported. GLAD is a single-step physical vapor deposition (PVD) technique used to fabricate porous nanostructured thin films. Using titanium dioxide, a transparent semiconductor with a high refractive index (n=2.4), the GLAD technique can be employed to fabricate samples with tailored nano-porosity, refractive index periodicities, and high Q-factor reflectance spectra. The OR-PAM in vivo images of chorioallantoic membrane (CAM) of 5-day chicken embryo model are demonstrated. The phantom study shows lateral resolution and signal-to-noise ratio better than 7 μm and 35 dB, respectively. The sensitive GLAD FPI allows photoacoustic imaging down to a few-nJ pulse energy. To the best of our knowledge, this is the first time that a FPI-based reflection mode optical resolution photoacoustic imaging technique is demonstrated for in vivo applications. PMID:25831330

  2. Stretching of red blood cells by optical tweezers quantified by digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Cardenas, Nelson; Yu, Lingfeng; Mohanty, Samarendra K.

    2011-03-01

    Red blood cells (RBC) possess unique viscoelastic characteristics which allow them to pass through capillaries narrower than their size. Measurement of viscoelastic property of cells (e.g. RBC) in low-force regime is of high significance as it represents conditions of membrane fluctuation in response to physiological conditions. Estimation of visco-elastic properties of RBC requires measurement of extent of deformation in RBC subjected to known force. Optical tweezers, being gentle and absolutely sterile, are emerging as the tool of choice for application of localized force on cells. However, stretching of RBC in very low force regime has not been quantified. Further, though deformations in transverse directions have been measured, vertical deformations due to stretching of cells cannot be quantified by classical microscopic images. Here, we report realization of offaxis digital holographic microscopy (DHM) for highly sensitive axial changes in RBC shape due to stretching by optical tweezers without attaching microscopic beads. The RBC was stretched in axial direction with nanometer precision by change of divergence of the trapping beam. The obtained deformation patterns were compared with the axial position of the tweezers focus. Since the pathophysiology of progression of diseases like malaria and cancer is reflected in the biophysical (both mechanical and material) properties of the cells, it is possible to identify the changes by simultaneous measurement of refractive index and elasticity using this approach.

  3. Fast optical-resolution photoacoustic microscopy using a 2-axis water-proofing MEMS scanner

    PubMed Central

    Kim, Jin Young; Lee, Changho; Park, Kyungjin; Lim, Geunbae; Kim, Chulhong

    2015-01-01

    Optical-resolution photoacoustic microscopy (OR-PAM) is a novel label-free microscopic imaging tool to provide in vivo optical absorbing contrasts. Specially, it is crucial to equip a real-time imaging capability without sacrificing high signal-to-noise ratios (SNRs) for identifying and tracking specific diseases in OR-PAM. Herein we demonstrate a 2-axis water-proofing MEMS scanner made of flexible PDMS. This flexible scanner results in a wide scanning range (9 × 4 mm2 in a transverse plane) and a fast imaging speed (5 B-scan images per second). Further, the MEMS scanner is fabricated in a compact footprint with a size of 15 × 15 × 15 mm3. More importantly, the scanning ability in water makes the MEMS scanner possible to confocally and simultaneously reflect both ultrasound and laser, and consequently we can maintain high SNRs. The lateral and axial resolutions of the OR-PAM system are 3.6 and 27.7 μm, respectively. We have successfully monitored the flow of carbon particles in vitro with a volumetric display frame rate of 0.14 Hz. Finally, we have successfully obtained in vivo PA images of microvasculatures in a mouse ear. It is expected that our compact and fast OR-PAM system can be significantly useful in both preclinical and clinical applications. PMID:25604654

  4. Methodology to set up accurate OPC model using optical CD metrology and atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Shim, Yeon-Ah; Kang, Jaehyun; Lee, Sang-Uk; Kim, Jeahee; Kim, Keeho

    2007-03-01

    For the 90nm node and beyond, smaller Critical Dimension(CD) control budget is required and the ways to control good CD uniformity are needed. Moreover Optical Proximity Correction(OPC) for the sub-90nm node demands more accurate wafer CD data in order to improve accuracy of OPC model. Scanning Electron Microscope (SEM) is the typical method for measuring CD until ArF process. However SEM can give serious attack such as shrinkage of Photo Resist(PR) by burning of weak chemical structure of ArF PR due to high energy electron beam. In fact about 5nm CD narrowing occur when we measure CD by using CD-SEM in ArF photo process. Optical CD Metrology(OCD) and Atomic Force Microscopy(AFM) has been considered to the method for measuring CD without attack of organic materials. Also the OCD and AFM measurement system have the merits of speed, easiness and accurate data. For model-based OPC, the model is generated using CD data of test patterns transferred onto the wafer. In this study we discuss to generate accurate OPC model using OCD and AFM measurement system.

  5. Single myelin fiber imaging in living rodents without labeling by deep optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Ben Arous, Juliette; Binding, Jonas; Léger, Jean-François; Casado, Mariano; Topilko, Piotr; Gigan, Sylvain; Claude Boccara, A.; Bourdieu, Laurent

    2011-11-01

    Myelin sheath disruption is responsible for multiple neuropathies in the central and peripheral nervous system. Myelin imaging has thus become an important diagnosis tool. However, in vivo imaging has been limited to either low-resolution techniques unable to resolve individual fibers or to low-penetration imaging of single fibers, which cannot provide quantitative information about large volumes of tissue, as required for diagnostic purposes. Here, we perform myelin imaging without labeling and at micron-scale resolution with >300-μm penetration depth on living rodents. This was achieved with a prototype [termed deep optical coherence microscopy (deep-OCM)] of a high-numerical aperture infrared full-field optical coherence microscope, which includes aberration correction for the compensation of refractive index mismatch and high-frame-rate interferometric measurements. We were able to measure the density of individual myelinated fibers in the rat cortex over a large volume of gray matter. In the peripheral nervous system, deep-OCM allows, after minor surgery, in situ imaging of single myelinated fibers over a large fraction of the sciatic nerve. This allows quantitative comparison of normal and Krox20 mutant mice, in which myelination in the peripheral nervous system is impaired. This opens promising perspectives for myelin chronic imaging in demyelinating diseases and for minimally invasive medical diagnosis.

  6. Modifications of the structure of the pericellular matrix measured via optical force probe microscopy

    NASA Astrophysics Data System (ADS)

    McLane, Louis; Kramer, Anthony; Chang, Patrick; Curtis, Jennifer

    2013-03-01

    The pericellular matrix is a large protein and polysaccharide rich polymer layer attached to the surface of many cells, and which often extends several microns out from the cell surface into the surrounding extracellular space. Here we study the intrinsic nature and modifications of the structure of the pericellular coat on rat chondrocytes with the use of optical force probe microscopy. Optical force probe studies allow us to make both dynamic force measurements as well as equilibrium force measurements throughout the coat. These force measurements are used to observe the structural change in the coat with the addition of exogenous aggrecan. Not only does addition of exogenous aggrecan dramatically swell our coat to well over twice in size, our analysis indicates that the addition of exogenous aggrecan decreases the mesh size throughout the coat. We speculate that the added aggrecan binds to available binding sites along the hyaluronan chain, both enlarging the coat's size as well as tightening up the opening within the coat. We further suggest that the available binding sites for the exogenous aggrecan are abundant in the outer edges of the coat, as both the dynamic and equilibrium forces in this region are changed. Here, both force measurements show that forces closest to the cell membrane remain relatively unchanged, while the forces in the outer region of the coat are increased. These results are consistent with those obtained with complementary measurements using quantitative particle exclusion assays.

  7. Mosaicing for fast wide-field-of-view optical resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Shao, Peng; Shi, Wei; Chee, Ryan K.; Forbrich, Alexander; Zemp, Roger J.

    2012-02-01

    The acquisition speed of previously reported mechanically-scanned Optical-Resolution Photoacoustic Microscopy (OR-PAM) systems has been limited by both laser pulse repetition rate and mechanical scanning speed. In this paper we introduce a mosaicing scheme wherein a grid of small sub-mm-scale field-of-view (FOV) patches are acquired in 0.5s per patch, and a 3-axis stepper-motor system is used to mechanically move the object to be imaged from patch-to-patch in less than 0.5s. Patch images are aligned and stitched to generate a large FOV image composite. This system retains the SNR-advantages of focused-transducer OR-PAM systems, and is a hybrid approach between optical-scanning and mechanical scanning. With this strategy we reduce the data acquisition time of previously reported large-FOV systems by a factor of around 23. SCID hairless mice are imaged. The wide-FOV, high-speed data acquisition OR-PAM system broadens the potential applications of the imaging modality.

  8. Determination of crystal grain orientations by optical microscopy at textured surfaces

    SciTech Connect

    Lausch, D.; Gläser, M.; Hagendorf, C.

    2013-11-21

    In this contribution, a new method to determine the crystal orientation with the example of chemical treated silicon wafers by means of optical microscopy has been demonstrated. The introduced procedure represents an easy method to obtain all relevant parameters to describe the crystal structure of the investigated material, i.e., the crystal grain orientation and the grain boundary character. The chemical treatment is a standard mono-texture for solar cells, well known in the solar industry. In general, this concept can also be applied to other crystalline materials, i.e., GaAs, SiC, etc., the only thing that needs to be adjusted is the texturing method to reveal specific crystal planes and the calculation model. In conclusion, an application of this method is shown with the example of the defect classification of recombination active defects in mc-Si solar cell. The introduced method demonstrates a simple and quick opportunity to improve the crystallization process and the quality of electronic devices by means of an optical microscope and a chemical treatment of the material.

  9. Integrated intravascular ultrasound and optical-resolution photoacoustic microscopy with a 1-mm-diameter catheter

    NASA Astrophysics Data System (ADS)

    Bai, Xiaosong; Gong, Xiaojing; Lin, Riqiang; Hau, William; Song, Liang

    2014-03-01

    Intravascular ultrasound (IVUS) plays a vital role in assessing the severity of atherosclerosis and has greatly enriched our knowledge on atherosclerotic plaques. However, it mainly reveals the structural information of plaques. In contrast, spectroscopic and molecular photoacoustic imaging can potentially improve plaque composition identification, inflammation detection, and ultimately the stratification of plaque vulnerability and risk. In this work, we developed an integrated intravascular ultrasound and optical-resolution photoacoustic microscopy (IVUS-PAM) system with a single catheter as small as 1 mm in diameter, comparable to that of existing clinical IVUS catheters. In addition, by using a GRIN lens to focus the excitation laser pulse, the system provides an optical-diffraction limited photoacoustic lateral resolution as fine as 19.6 micrometers, ~10-fold finer than that of conventional intravascular photoacoustic imaging and existing IVUS technology. The system employs a custom-made miniaturized single-element ultrasonic transducer with a dimension of ~0.5 mm, a centre frequency of ~40 MHz, and a fractional bandwidth of ~60%. The IVUS-PAM can simultaneously acquire co-registered IVUS images with an axial resolution of ~40 micrometers and a lateral resolution of ~200 micrometers. In the future, IVUS-PAM may open up new opportunities for improved high-resolution vulnerable plaque imaging and image-guided stent deployment.

  10. Implementation of adaptive optics in fluorescent microscopy using wavefront sensing and correction

    NASA Astrophysics Data System (ADS)

    Azucena, Oscar; Crest, Justin; Cao, Jian; Sullivan, William; Kner, Peter; Gavel, Donald; Dillon, Daren; Olivier, Scot; Kubby, Joel

    2010-02-01

    Adaptive optics (AO) improves the quality of astronomical imaging systems by using real time measurement of the turbulent medium in the optical path using a guide star (natural or artificial) as a point source reference beacon [1]. AO has also been applied to vision science to improve the view of the human eye. This paper will address our current research focused on the improvement of fluorescent microscopy for biological imaging utilizing current AO technology. A Shack-Hartmann wavefront sensor (SHWS) is used to measure the aberration introduced by a Drosophila Melanogaster embryo with an implanted 1 micron fluorescent bead that serves as a point source reference beacon. Previous measurements of the wavefront aberrations have found an average peak-to-valley and root-mean-square (RMS) wavefront error of 0.77 micrometers and 0.15 micrometers, respectively. Measurements of the Zernike coefficients indicated that the correction of the first 14 Zernike coefficients is sufficient to correct the aberrations we measured. Here we show that a MEMS deformable mirror with 3.5 microns of stroke and 140 actuators is sufficient to correct these aberrations. The design, assembly and initial results for the use of a MEMS deformable mirror, SHWS and implanted fluorescent reference beacon for wavefront correction are discussed.

  11. Dye-enhanced reflectance and fluorescence confocal microscopy as an optical pathology tool

    NASA Astrophysics Data System (ADS)

    Yaroslavsky, Anna N.; Salomatina, Elena; Novak, John; Amat-Roldan, Ivan; Castano, Ana; Hamblin, Michael

    2006-02-01

    Early detection and precise excision of neoplasms are imperative requirements for successful cancer treatment. In this study we evaluated the use of dye-enhanced confocal microscopy as an optical pathology tool in the ex vivo trial with fresh thick non-melanoma skin cancer excisions and in vivo trial with B16F10 melanoma cancer in mice. For the experiments the tumors were rapidly stained using aqueous solutions of either toluidine blue or methylene blue and imaged using multimodal confocal microscope. Reflectance images were acquired at the wavelengths of 630nm and 650 nm. Fluorescence was excited at 630 nm and 650 nm. Fluorescence emission was registered in the range between 680 nm and 710 nm. The images were compared to the corresponding en face frozen H&E sections. The results of the study indicate confocal images of stained cancerous tissue closely resemble corresponding H&E sections both in vivo and in vitro. This remarkable similarity enables interpretation of confocal images in a manner similar to that of histopathology. The developed technique may provide an efficient real-time optical tool for detecting skin pathology.

  12. Lab on chip optical imaging of biological sample by quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Memmolo, P.; Miccio, L.; Merola, F.; Gennari, O.; Mugnano, M.; Netti, P. A.; Ferraro, P.

    2015-03-01

    Quantitative imaging and three dimensional (3D) morphometric analysis of flowing and not-adherent cells is an important aspect for diagnostic purposes at Lab on Chip scale. Diagnostics tools need to be quantitative, label-free and, as much as possible, accurate. In recent years digital holography (DH) has been improved to be considered as suitable diagnostic method in several research field. In this paper we demonstrate that DH can be used for retrieving 3D morphometric data for sorting and diagnosis aims. Several techniques exist for 3D morphological study as optical coherent tomography and confocal microscopy, but they are not the best choice in case of dynamic events as flowing samples. Recently, a DH approach, based on shape from silhouette algorithm (SFS), has been developed for 3D shape display and calculation of cells biovolume. Such approach, adopted in combination with holographic optical tweezers (HOT) was successfully applied to cells with convex shape. Unfortunately, it's limited to cells with convex surface as sperm cells or diatoms. Here, we demonstrate an improvement of such procedure. By decoupling thickness information from refractive index ones and combining this with SFS analysis, 3D shape of concave cells is obtained. Specifically, the topography contour map is computed and used to adjust the 3D shape retrieved by the SFS algorithm. We prove the new procedure for healthy red blood cells having a concave surface in their central region. Experimental results are compared with theoretical model.

  13. Combined optical coherence phase microscopy and impedance sensing measurements of differentiating adipose derived stem cells

    NASA Astrophysics Data System (ADS)

    Bagnaninchi, P. O.

    2010-02-01

    There is a growing interest in monitoring differentiating stem cells in 2D culture without the use of labelling agents. In this study we explore the feasibility of a multimodality method that combines impedance sensing (IS) and optical coherence phase microscopy (OCPM) to monitor the main biological events associated with adipose derived stem cells differentiation into different lineages. Adipose derived stem cells were cultured in Mesenpro RS medium on gold electrode arrays. The system (ECIS, Applied biophysics) is connected to a lock-in amplifier controlled by a computer, and the complex impedance is derived from the in phase and out of phase voltages. Multi-frequency measurements spanning from 500Hz to 100 kHz are recorded every 2 minutes. The Optical coherence phase microscope is build around a Thorlabs engine (930nm FWHM: 90nm) and connected to a custom build microscope probe. The IS and OCPM were successfully integrated. The electrode area (250um) was imaged with a lateral resolution of 1.5um during impedance measurements. Impedance sensing gave an average measurement of differentiation, as a change in impedance over the electrode area, whereas OCPM provides additional information on the cellular events occurring on top of the electrode. The information retrieved from OCPM will feed a mathematical model correlating cellular differentiation and impedance variation. In this study we have demonstrated the feasibility of integrating two non-invasive monitoring techniques that will be instrumental in designing stem cell based screening assays.

  14. Energy Landscape of Alginate-Epimerase Interactions Assessed by Optical Tweezers and Atomic Force Microscopy

    PubMed Central

    Håti, Armend Gazmeno; Aachmann, Finn Lillelund; Stokke, Bjørn Torger; Skjåk-Bræk, Gudmund; Sletmoen, Marit

    2015-01-01

    Mannuronan C-5 epimerases are a family of enzymes that catalyze epimerization of alginates at the polymer level. This group of enzymes thus enables the tailor-making of various alginate residue sequences to attain various functional properties, e.g. viscosity, gelation and ion binding. Here, the interactions between epimerases AlgE4 and AlgE6 and alginate substrates as well as epimerization products were determined. The interactions of the various epimerase–polysaccharide pairs were determined over an extended range of force loading rates by the combined use of optical tweezers and atomic force microscopy. When studying systems that in nature are not subjected to external forces the access to observations obtained at low loading rates, as provided by optical tweezers, is a great advantage since the low loading rate region for these systems reflect the properties of the rate limiting energy barrier. The AlgE epimerases have a modular structure comprising both A and R modules, and the role of each of these modules in the epimerization process were examined through studies of the A- module of AlgE6, AlgE6A. Dynamic strength spectra obtained through combination of atomic force microscopy and the optical tweezers revealed the existence of two energy barriers in the alginate-epimerase complexes, of which one was not revealed in previous AFM based studies of these complexes. Furthermore, based on these spectra estimates of the locations of energy transition states (xβ), lifetimes in the absence of external perturbation (τ0) and free energies (ΔG#) were determined for the different epimerase–alginate complexes. This is the first determination of ΔG# for these complexes. The values determined were up to 8 kBT for the outer barrier, and smaller values for the inner barriers. The size of the free energies determined are consistent with the interpretation that the enzyme and substrate are thus not tightly locked at all times but are able to relocate. Together with the

  15. Optical sectioning microscopy using two-frame structured illumination and Hilbert-Huang data processing

    NASA Astrophysics Data System (ADS)

    Trusiak, M.; Patorski, K.; Tkaczyk, T.

    2014-12-01

    We propose a fast, simple and experimentally robust method for reconstructing background-rejected optically-sectioned microscopic images using two-shot structured illumination approach. Innovative data demodulation technique requires two grid-illumination images mutually phase shifted by π (half a grid period) but precise phase displacement value is not critical. Upon subtraction of the two frames the input pattern with increased grid modulation is computed. The proposed demodulation procedure comprises: (1) two-dimensional data processing based on the enhanced, fast empirical mode decomposition (EFEMD) method for the object spatial frequency selection (noise reduction and bias term removal), and (2) calculating high contrast optically-sectioned image using the two-dimensional spiral Hilbert transform (HS). The proposed algorithm effectiveness is compared with the results obtained for the same input data using conventional structured-illumination (SIM) and HiLo microscopy methods. The input data were collected for studying highly scattering tissue samples in reflectance mode. In comparison with the conventional three-frame SIM technique we need one frame less and no stringent requirement on the exact phase-shift between recorded frames is imposed. The HiLo algorithm outcome is strongly dependent on the set of parameters chosen manually by the operator (cut-off frequencies for low-pass and high-pass filtering and η parameter value for optically-sectioned image reconstruction) whereas the proposed method is parameter-free. Moreover very short processing time required to efficiently demodulate the input pattern predestines proposed method for real-time in-vivo studies. Current implementation completes full processing in 0.25s using medium class PC (Inter i7 2,1 GHz processor and 8 GB RAM). Simple modification employed to extract only first two BIMFs with fixed filter window size results in reducing the computing time to 0.11s (8 frames/s).

  16. Mirage effect spectrometry and light profile microscopy: Two views of an optical depth profile (abstract)

    NASA Astrophysics Data System (ADS)

    Power, J. F.; Fu, S. W.; Nepotchatykh, O. V.

    2003-01-01

    Photothermal depth profiling techniques are well adapted for the inspection of optically absorbing features on the length scale of 1-100 μm in a variety of media. However, the depth profiling mechanism intrinsic to thermal wave imaging is inherently ill posed [J. F. Power, AIP Conf. Proc. 463, 3 (1999)], and suffers obvious disadvantages such as sensitivity to experimental errors (requiring regularization) and subsurface broadening of the regularized depth profiles. Recently, through the introduction of light profile microscopy (LPM) an alternate method of optical inspection was made available for depth profiling optically absorbing, scattering, and luminescent structures on this length scale [J. F. Power and S. W. Fu, Appl. Spectros. 53, 1507 (1999); J. F. Power and S. W. Fu, U.S. Patent Pending]. LPM inspects a thin film under test by directing a laser beam through the material along the depth axis, parallel to a polished cross-sectional viewing surface. Luminescence and elastic scatter excited in the beam volume is imaged by a microscope aligned orthogonal to the beam axis. The images obtained by this method showed striking depth contrast in a variety of materials with subsurface interfaces and depth variations of luminescence yield. When implemented in dual beam mode [J. F. Power and S. W. Fu, U.S. Patent Pending; J. F. Power and S. W. Fu, (unpublished)] with an associated mathematical method, LPM may be used to quantitatively resolve depth variable optical absorption from light scattering and luminescence efficiency. In contrast to photothermal methods, the LPM technique is well posed. LPM was evaluated in tandem with mirage effect spectrometry (in normal deflection mode with bicell detection) [J. F. Power, S. W. Fu, and M. A. Schweitzer, Appl. Spectros. 54, 110 (2000)], to determine the effective use of each technique in analysis problems on complex materials. This study used samples with known depth variations of optical properties including homogeneous

  17. Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a review

    NASA Astrophysics Data System (ADS)

    Sednev, Maksim V.; Belov, Vladimir N.; Hell, Stefan W.

    2015-12-01

    The review deals with commercially available organic dyes possessing large Stokes shifts and their applications as fluorescent labels in optical microscopy based on stimulated emission depletion (STED). STED microscopy breaks Abbe’s diffraction barrier and provides optical resolution beyond the diffraction limit. STED microscopy is non-invasive and requires photostable fluorescent markers attached to biomolecules or other objects of interest. Up to now, in most biology-related STED experiments, bright and photoresistant dyes with small Stokes shifts of 20-40 nm were used. The rapid progress in STED microscopy showed that organic fluorophores possessing large Stokes shifts are indispensable in multi-color super-resolution techniques. The ultimate result of the imaging relies on the optimal combination of a dye, the bio-conjugation procedure and the performance of the optical microscope. Modern bioconjugation methods, basics of STED microscopy, as well as structures and spectral properties of the presently available fluorescent markers are reviewed and discussed. In particular, the spectral properties of the commercial dyes are tabulated and correlated with the available depletion wavelengths found in STED microscopes produced by LEICA Microsytems, Abberior Instruments and Picoquant GmbH.

  18. Adaptive and Background-Aware GAL4 Expression Enhancement of Co-registered Confocal Microscopy Images.

    PubMed

    Trapp, Martin; Schulze, Florian; Novikov, Alexey A; Tirian, Laszlo; J Dickson, Barry; Bühler, Katja

    2016-04-01

    GAL4 gene expression imaging using confocal microscopy is a common and powerful technique used to study the nervous system of a model organism such as Drosophila melanogaster. Recent research projects focused on high throughput screenings of thousands of different driver lines, resulting in large image databases. The amount of data generated makes manual assessment tedious or even impossible. The first and most important step in any automatic image processing and data extraction pipeline is to enhance areas with relevant signal. However, data acquired via high throughput imaging tends to be less then ideal for this task, often showing high amounts of background signal. Furthermore, neuronal structures and in particular thin and elongated projections with a weak staining signal are easily lost. In this paper we present a method for enhancing the relevant signal by utilizing a Hessian-based filter to augment thin and weak tube-like structures in the image. To get optimal results, we present a novel adaptive background-aware enhancement filter parametrized with the local background intensity, which is estimated based on a common background model. We also integrate recent research on adaptive image enhancement into our approach, allowing us to propose an effective solution for known problems present in confocal microscopy images. We provide an evaluation based on annotated image data and compare our results against current state-of-the-art algorithms. The results show that our algorithm clearly outperforms the existing solutions. PMID:26743993

  19. Optical Coherence Tomography Angiography in Mice: Comparison with Confocal Scanning Laser Microscopy and Fluorescein Angiography

    PubMed Central

    Giannakaki-Zimmermann, Helena; Kokona, Despina; Wolf, Sebastian; Ebneter, Andreas; Zinkernagel, Martin S.

    2016-01-01

    Purpose Optical coherence tomography angiography (OCT-A) allows noninvasive visualization of retinal vessels in vivo. OCT-A was used to characterize the vascular network of the mouse retina and was compared with fluorescein angiography (FA) and histology. Methods In the present study, OCT-A based on a Heidelberg Engineering Spectralis system was used to investigate the vascular network in mice. Data was compared with FA and confocal microscopy of flat-mount histology stained with isolectin IB4. For quantitative analysis the National Cancer Institute's AngioTool software was used. Vessel density, the number of vessel junctions, and endpoints were measured and compared between the imaging modalities. Results The configuration of the superficial capillary network was comparable with OCT-A and flat-mount histology in BALBc mice. However, vessel density and the number of vessel junctions per region of interest (P = 0.0161 and P = 0.0015, respectively) in the deep vascular network of BALBc mice measured by OCT-A was significantly higher than with flat-mount histology. In C3A.Cg-Pde6b+Prph2Rd2/J mice, where the deep capillary plexus is absent, analysis of the superficial network provided similar results for all three imaging modalities. Conclusion OCT-A is a helpful imaging tool for noninvasive, in vivo imaging of the vascular plexus in mice. It may offer advantages over FA and confocal microscopy especially for imaging the deep vascular plexus. Translational Relevance The present study shows that OCT-A can be employed for small animal imaging to assess the vascular network and offers advantages over flat-mount histology and FA. PMID:27570710

  20. Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

    PubMed Central

    Sivaguru, Mayandi; Fried, Glenn A.; Miller, Carly A. H.; Fouke, Bruce W.

    2014-01-01

    An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation, (2) superior optical sectioning ability, and (3) minimal light absorption and scattering, while still permitting deep tissue imaging. PMID:25226350

  1. Optical analysis of nanomaterial-cell interactions: flow cytometry and digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Mues, Sarah; Antunovic, Jan; Ossig, Rainer; Kemper, Björn; Schnekenburger, Jürgen

    2015-05-01

    The in vitro cytotoxicity assessment of engineered nanoparticles commonly involves the measurement of different endpoints like the formation of reactive oxygen species, cell viability or cell death. Usually these parameters are determined by optical readouts of enzymatically converted substrates that often interfere with the tested nanomaterials. Using cell viability (WST-8) and cell death (LDH) as parameter we have initially investigated the toxic effects of spherical (NM 300) and rod shaped (NM 302) silver nanomaterials with a matrix of four cell lines representing different functions: lung and kidney epithelial cells, macrophages and fibroblasts. In addition, we have used a label-free flow cytometer configuration to investigate interactions of particles and macrophages by side scatter signal analysis. Finally, we explored digital holographic microscopy (DHM) for multimodal label-free analysis of nanomaterial toxicity. Quantitative DHM phase images were analyzed for cell thickness, volume, density, dry mass and refractive index. We could demonstrate that silver spheres lead to more cytotoxic effects than rods in all four examined cell lines and both assay. Exemplarily a dose dependent interaction increase of cells with NM 300 and NM 302 analyzed by flow cytometry is shown. Furthermore, we found that the refractive index of cells is influenced by incubation with NM 300 in a decreasing manner. A 24 hours time-lapse measurement revealed a dose dependent decrease of dry mass and surface area development indicating reduced cell viability and cell death. Our results demonstrate digital holographic microscopy and flow cytometry as valuable label-free tools for nanomaterial toxicity and cell interaction studies.

  2. Investigation into local cell mechanics by atomic force microscopy mapping and optical tweezer vertical indentation.

    PubMed

    Coceano, G; Yousafzai, M S; Ma, W; Ndoye, F; Venturelli, L; Hussain, I; Bonin, S; Niemela, J; Scoles, G; Cojoc, D; Ferrari, E

    2016-02-12

    Investigating the mechanical properties of cells could reveal a potential source of label-free markers of cancer progression, based on measurable viscoelastic parameters. The Young's modulus has proved to be the most thoroughly studied so far, however, even for the same cell type, the elastic modulus reported in different studies spans a wide range of values, mainly due to the application of different experimental conditions. This complicates the reliable use of elasticity for the mechanical phenotyping of cells. Here we combine two complementary techniques, atomic force microscopy (AFM) and optical tweezer microscopy (OTM), providing a comprehensive mechanical comparison of three human breast cell lines: normal myoepithelial (HBL-100), luminal breast cancer (MCF-7) and basal breast cancer (MDA-MB-231) cells. The elastic modulus was measured locally by AFM and OTM on single cells, using similar indentation approaches but different measurement parameters. Peak force tapping AFM was employed at nanonewton forces and high loading rates to draw a viscoelastic map of each cell and the results indicated that the region on top of the nucleus provided the most meaningful results. OTM was employed at those locations at piconewton forces and low loading rates, to measure the elastic modulus in a real elastic regime and rule out the contribution of viscous forces typical of AFM. When measured by either AFM or OTM, the cell lines' elasticity trend was similar for the aggressive MDA-MB-231 cells, which were found to be significantly softer than the other two cell types in both measurements. However, when comparing HBL-100 and MCF-7 cells, we found significant differences only when using OTM. PMID:26683826

  3. Investigation into local cell mechanics by atomic force microscopy mapping and optical tweezer vertical indentation

    NASA Astrophysics Data System (ADS)

    Coceano, G.; Yousafzai, M. S.; Ma, W.; Ndoye, F.; Venturelli, L.; Hussain, I.; Bonin, S.; Niemela, J.; Scoles, G.; Cojoc, D.; Ferrari, E.

    2016-02-01

    Investigating the mechanical properties of cells could reveal a potential source of label-free markers of cancer progression, based on measurable viscoelastic parameters. The Young’s modulus has proved to be the most thoroughly studied so far, however, even for the same cell type, the elastic modulus reported in different studies spans a wide range of values, mainly due to the application of different experimental conditions. This complicates the reliable use of elasticity for the mechanical phenotyping of cells. Here we combine two complementary techniques, atomic force microscopy (AFM) and optical tweezer microscopy (OTM), providing a comprehensive mechanical comparison of three human breast cell lines: normal myoepithelial (HBL-100), luminal breast cancer (MCF-7) and basal breast cancer (MDA-MB-231) cells. The elastic modulus was measured locally by AFM and OTM on single cells, using similar indentation approaches but different measurement parameters. Peak force tapping AFM was employed at nanonewton forces and high loading rates to draw a viscoelastic map of each cell and the results indicated that the region on top of the nucleus provided the most meaningful results. OTM was employed at those locations at piconewton forces and low loading rates, to measure the elastic modulus in a real elastic regime and rule out the contribution of viscous forces typical of AFM. When measured by either AFM or OTM, the cell lines’ elasticity trend was similar for the aggressive MDA-MB-231 cells, which were found to be significantly softer than the other two cell types in both measurements. However, when comparing HBL-100 and MCF-7 cells, we found significant differences only when using OTM.

  4. Depth-resolved Optical Imaging and Microscopy of Vascular Compartment Dynamics During Somatosensory Stimulation

    PubMed Central

    Hillman, Elizabeth M. C.; Devor, Anna; Bouchard, Matthew; Dunn, Andrew K.; Krauss, GW; Skoch, Jesse; Bacskai, Brian J.; Dale, Anders M.; Boas, David A.

    2007-01-01

    The cortical hemodynamic response to somatosensory stimulus is investigated at the level of individual vascular compartments using both depth-resolved optical imaging and in-vivo two-photon microscopy. We utilize a new imaging and spatiotemporal analysis approach that exploits the different characteristic dynamics of responding arteries, arterioles, capillaries and veins to isolate their three-dimensional spatial extent within the cortex. This spatial delineation is validated using vascular casts. Temporal delineation is supported by in-vivo two-photon microscopy of the temporal dynamics and vascular mechanisms of the arteriolar and venous responses. Using these techniques we have been able to characterize the roles of the different vascular compartments in generating and controlling the hemodynamic response to somatosensory stimulus. We find that changes in arteriolar total hemoglobin concentration agree well with arteriolar dilation dynamics, which in turn correspond closely with changes in venous blood flow. For four-second stimuli, we see only small changes in venous hemoglobin concentration, and do not detect measurable dilation or ballooning in the veins. Instead, we see significant evidence of capillary hyperemia. We compare our findings to historical observations of the composite hemodynamic response from other modalities including functional magnetic resonance imaging. Implications of our results are discussed with respect to mathematical models of cortical hemodynamics, and to current theories on the mechanisms underlying neurovascular coupling. We also conclude that our spatiotemporal analysis approach is capable of isolating and localizing signals from the capillary bed local to neuronal activation, and holds promise for improving the specificity of other hemodynamic imaging modalities. PMID:17222567

  5. Characterization of Si3N4/SiO2 optical channel waveguides by photon scanning tunneling microscopy

    NASA Technical Reports Server (NTRS)

    Wang, Yan; Chudgar, Mona H.; Jackson, Howard E.; Miller, Jeffrey S.; De Brabander, Gregory N.; Boyd, Joseph T.

    1993-01-01

    Photon scanning tunneling microscopy (PSTM) is used to characterize Si3N4/Si02 optical channel waveguides being used for integrated optical-micromechanical sensors. PSTM utilizes an optical fiber tapered to a fine point which is piezoelectrically positioned to measure the decay of the evanescent field intensity associated with the waveguide propagating mode. Evanescent field decays are recorded for both ridge channel waveguides and planar waveguide regions. Values for the local effective refractive index are calculated from the data for both polarizations and compared to model calculations.

  6. High-sensitivity piezoelectric tube sensor for shear-force detection in scanning near-field optical microscopy

    NASA Astrophysics Data System (ADS)

    Lindfors, K.; Kapulainen, M.; Ryytty, P.; Kaivola, M.

    2004-11-01

    An easy-to-implement non-optical shear-force detection setup for tip-sample distance regulation in scanning near-field optical microscopy is demonstrated. The detection method is based on attaching the near-field probe to a piezoelectric tube resulting in excellent mechanical contact between tip and detector. The main advantages of the method are good signal-to-background contrast and thus potential for high sensitivity. The method is demonstrated by obtaining approach curves of silicon surfaces. The suitability for optical experiments is further shown by measuring the near-field intensity distribution of the emission of a semiconductor laser.

  7. Back-etch method for plan view transmission electron microscopy sample preparation of optically opaque films.

    PubMed

    Yao, Bo; Coffey, Kevin R

    2008-04-01

    Back-etch methods have been widely used to prepare plan view transmission electron microscopy (TEM) samples of thin films on membranes by removal of the Si substrate below the membrane by backside etching. The conventional means to determine when to stop the etch process is to observe the color of the light transmitted through the sample, which is sensitive to the remaining Si thickness. However, most metallic films thicker than 75 nm are opaque, and there is no detectable color change prior to film perforation. In this paper, a back-etch method based on the observation of an abrupt change of optical reflection contrast is introduced as a means to determine the etch endpoint to prepare TEM samples for these films. As the acid etchant removes the Si substrate material a rough interface is generated. This interface becomes a relatively smooth and featureless region when the etchant reaches the membrane (film/SiO2). This featureless region is caused by the mirror reflection of the film plane (film/SiO2 interface) through the optically transparent SiO2 layer. The lower etch rate of SiO2 (compared with Si) gives the operator enough time to stop the etching without perforating the film. A clear view of the morphology and control of Si roughness during etching are critical to this method, which are discussed in detail. The procedures of mounting wax removal and sample rinsing are also described in detail, as during these steps damage to the membrane may easily occur without appropriate consideration. As examples, the preparation of 100-nm-thick Fe-based amorphous alloy thin film and 160-nm-thick Cu-thin film samples for TEM imaging is described. PMID:18227137

  8. Optical mapping of conduction in early embryonic quail hearts with light-sheet microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Ma, Pei; Gu, Shi; Wang, Yves T.; Jenkins, Michael W.; Rollins, Andrew M.

    2016-03-01

    Optical mapping (OM) using fluorescent voltage-sensitive dyes (VSD) to measure membrane potential is currently the most effective method for electrophysiology studies in early embryonic hearts due to its noninvasiveness and large field-of-view. Conventional OM acquires bright-field images, collecting signals that are integrated in depth and projected onto a 2D plane, not capturing the 3D structure of the sample. Early embryonic hearts, especially at looping stages, have a complicated, tubular geometry. Therefore, conventional OM cannot provide a full picture of the electrical conduction circumferentially around the heart, and may result in incomplete and inaccurate measurements. Here, we demonstrate OM of Hamburger and Hamilton stage 14 embryonic quail hearts using a new commercially-available VSD, Fluovolt, and depth sectioning using a custom built light-sheet microscopy system. Axial and lateral resolution of the system is 14µm and 8µm respectively. For OM imaging, the field-of-view was set to 900µm×900µm to cover the entire heart. 2D over time OM image sets at multiple cross-sections through the looping-stage heart were recorded. The shapes of both atrial and ventricular action potentials acquired were consistent with previous reports using conventional VSD (di-4-ANNEPS). With Fluovolt, signal-to-noise ratio (SNR) is improved significantly by a factor of 2-10 (compared with di-4-ANNEPS) enabling light-sheet OM, which intrinsically has lower SNR due to smaller sampling volumes. Electrophysiologic parameters are rate dependent. Optical pacing was successfully integrated into the system to ensure heart rate consistency. This will also enable accurately gated reconstruction of full four dimensional conduction maps and 3D conduction velocity measurements.

  9. Towards non-invasive 3D hepatotoxicity assays with optical coherence phase microscopy

    NASA Astrophysics Data System (ADS)

    Nelson, Leonard J.; Koulovasilopoulos, Andreas; Treskes, Philipp; Hayes, Peter C.; Plevris, John N.; Bagnaninchi, Pierre O.

    2015-03-01

    Three-dimensional tissue-engineered models are increasingly recognised as more physiologically-relevant than standard 2D cell culture for pre-clinical drug toxicity testing. However, many types of conventional toxicity assays are incompatible with dense 3D tissues. This study investigated the use of optical coherence phase microscopy (OCPM) as a novel approach to assess cell death in 3D tissue culture. For 3D micro-spheroid formation Human hepatic C3A cells were encapsulated in hyaluronic acid gels and cultured in 100μl MEME/10%FBS in 96-well plates. After spheroid formation the 3D liver constructs were exposed to acetaminophen on culture day 8. Acetaminophen hepatotoxicity in 3D cultures was evaluated using standard biochemical assays. An inverted OCPM in common path configuration was developed with a Callisto OCT engine (Thorlabs), centred at 930nm and a custom scanning head. Intensity data were used to perform in-depth microstructural imaging. In addition, phase fluctuations were measured by collecting several successive B scans at the same location, and statistics on the first time derivative of the phase, i.e. time fluctuations, were analysed over the acquisition time interval to retrieve overall cell viability. OCPM intensity (cell cluster size) and phase fluctuation statistics were directly compared with biochemical assays. In this study, we investigated optical coherence phase tomography to assess cell death in a 3d liver model after exposure to a prototypical hepatotoxin, acetaminophen. We showed that OCPM has the potential to assess noninvasively and label-free drug toxicity in 3D tissue models.

  10. All-optical photoacoustic microscopy (AOPAM) system for remote characterization of biological tissues

    NASA Astrophysics Data System (ADS)

    Sampathkumar, Ashwin; Chitnis, Parag V.; Silverman, Ronald H.

    2014-03-01

    Conventional photoacoustic microscopy (PAM) employs light pulses to produce a photoacoustic (PA) effect and detects the resulting acoustic waves using an ultrasound transducer acoustically coupled to the target. The resolution of conventional PAM is limited by the sensitivity and bandwidth of the ultrasound transducer. We investigated a versatile, all-optical PAM (AOPAM) system for characterizing in vivo as well as ex vivo biological specimens. The system employs non-contact interferometric detection of PA signals that overcomes limitations of conventional PAM. A 532-nm pump laser with a pulse duration of 5 ns excites the PA effect in tissue. Resulting acoustic waves produce surface displacements that are sensed using a 532-nm continuous-wave (CW) probe laser in a Michelson interferometer with a 1- GHz bandwidth. The pump and probe beams are coaxially focused using a 50X objective giving a diffraction-limited spot size of 0.48 μm. The phase-encoded probe beam is demodulated using homodyne methods. The detected timedomain signal is time reversed using k-space wave-propagation methods to produce a spatial distribution of PA sources in the target tissue. A minimum surface-displacement sensitivity of 0.19 pm was measured. PA-induced surface displacements are very small; therefore, they impose stringent detection requirements and determine the feasibility of implementing an all-optical PAM in biomedical applications. 3D PA images of ex vivo porcine retina specimens were generated successfully. We believe the AOPAM system potentially is well suited for assessing retinal diseases and other near-surface biomedical applications such as sectionless histology and evaluation of skin burns and pressure or friction ulcers.

  11. Characterizing deep optical-sectioning microscopy performance with scattering phantoms and numerical simulations

    NASA Astrophysics Data System (ADS)

    Liu, Jonathan T. C.; Mandella, Michael J.; Kino, Gordon S.; Contag, Christopher H.

    2010-02-01

    Microscopes are being developed for use in living animals, and even humans, to image microanatomical changes and molecular markers that are associated with disease. Phantoms that can be used to evaluate the performance characteristics of these systems have not been well described or standardized. We have been developing the tools to evaluate a dual-axis confocal (DAC) microscope design to optimize the features required for in vivo diagnosic imaging, and these may have features that are useful for evaluation of other such devices. We have performed diffraction-theory modeling, Monte-Carlo scattering simulations, reflectance experiments in tissue phantoms, and tissue-imaging validations. First, we determined how scattering from tissue deteriorates the diffraction-limited transverse and vertical responses in reflectance DAC imaging. Specifically, the vertical and transverse responses of the DAC to a plane reflector and a knife edge, respectively, were measured at various depths in an Intralipid scattering phantom. Comparisons were made with both diffraction-theory and Monte-Carlo scattering simulations. Secondly, as a practical demonstration of deep-tissue fluorescence microscopy, three-dimensional fluorescence images were obtained in thick human biopsy samples. These results demonstrate that the efficient rejection of scattered light in a DAC microscope enables deep optical sectioning in tissue. Finally, we will discuss our needs and plans for similar tissue-phantom experiments to validate the performance of multimodal optical- and ultrasound-imaging platforms under development. As devices are developed for the imaging of epithelial surfaces and substructures, standardized phantoms that represent the multilayered anatomical features of these tissues will need to be developed.

  12. Fluorescence Microscopy

    PubMed Central

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

    2016-01-01

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

  13. A Circadian Clock Gene, Cry, Affects Heart Morphogenesis and Function in Drosophila as Revealed by Optical Coherence Microscopy

    PubMed Central

    Zeng, Xianxu; Tate, Rebecca E.; McKee, Mary L.; Capen, Diane E.; Zhang, Zhan; Tanzi, Rudolph E.; Zhou, Chao

    2015-01-01

    Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal in vivo are poorly understood. The Drosophila cryptochrome (dCry) is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of dCry showed a significant increase (>100-fold) in Drosophila during the pupa and adult stages. In this study, we utilized an ultrahigh resolution optical coherence microscopy (OCM) system to perform non-invasive and longitudinal analysis of functional and morphological changes in the Drosophila heart throughout its post-embryonic lifecycle for the first time. The Drosophila heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR) and cardiac activity period (CAP) of Drosophila showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time) OCM images, cardiac structural and functional parameters of Drosophila at different developmental stages were quantitatively determined. In order to study the functional role of dCry on Drosophila heart development, we silenced dCry by RNAi in the Drosophila heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays an essential

  14. A Circadian Clock Gene, Cry, Affects Heart Morphogenesis and Function in Drosophila as Revealed by Optical Coherence Microscopy.

    PubMed

    Alex, Aneesh; Li, Airong; Zeng, Xianxu; Tate, Rebecca E; McKee, Mary L; Capen, Diane E; Zhang, Zhan; Tanzi, Rudolph E; Zhou, Chao

    2015-01-01

    Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal in vivo are poorly understood. The Drosophila cryptochrome (dCry) is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of dCry showed a significant increase (>100-fold) in Drosophila during the pupa and adult stages. In this study, we utilized an ultrahigh resolution optical coherence microscopy (OCM) system to perform non-invasive and longitudinal analysis of functional and morphological changes in the Drosophila heart throughout its post-embryonic lifecycle for the first time. The Drosophila heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR) and cardiac activity period (CAP) of Drosophila showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time) OCM images, cardiac structural and functional parameters of Drosophila at different developmental stages were quantitatively determined. In order to study the functional role of dCry on Drosophila heart development, we silenced dCry by RNAi in the Drosophila heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays an essential

  15. Exploring the limits of optical microscopy: live cell and superresolution fluorescence microscopy of HIV-1 Transfer Between T lymphocytes Across the Virological Synapse

    NASA Astrophysics Data System (ADS)

    McNerney, Gregory Paul

    Human immunodeficiency virus 1 (HIV-1) is a human retrovirus that efficiently, albeit gradually, overruns the immune system. An already infected T lymphocyte can latch onto another T lymphocyte whereby creating a virological synapse (VS); this junction drives viral assembly and transfer to the target cell in batches in an efficient, protective manor. My Ph.D. doctoral thesis focused on studying this transmission mechanism using advanced optical imaging modalities and the fully infectious fluorescent clone HIV Gag-iGFP. T lymphocytes are non-adherent cells (˜10 um thick) and the viral transmission process is fairly dynamic, hence we employed a custom spinning disk confocal microscope that revealed many interesting characteristics of this cooperative event. This methodology has low throughput as cell contact and transfer is at random. Optical tweezers was then added to the microscope to directly initiate cell contact at will. To assess when viral maturation occurs post-transfer, an optical assay based off of Forster resonance energy transfer was developed to monitor maturation. Structured illumination microscopy was further used to image the process at higher resolution and it showed that viral particles are not entering existing degradative compartments. Non-HIV-1 applications of the optical technologies are also reviewed.

  16. Molecular organization of the desmosome as revealed by direct stochastic optical reconstruction microscopy.

    PubMed

    Stahley, Sara N; Bartle, Emily I; Atkinson, Claire E; Kowalczyk, Andrew P; Mattheyses, Alexa L

    2016-08-01

    Desmosomes are macromolecular junctions responsible for providing strong cell-cell adhesion. Because of their size and molecular complexity, the precise ultrastructural organization of desmosomes is challenging to study. Here, we used direct stochastic optical reconstruction microscopy (dSTORM) to resolve individual plaque pairs for inner and outer dense plaque proteins. Analysis methods based on desmosomal mirror symmetry were developed to measure plaque-to-plaque distances and create an integrated map. We quantified the organization of desmoglein 3, plakoglobin and desmoplakin (N-terminal, rod and C-terminal domains) in primary human keratinocytes. Longer desmosome lengths correlated with increasing plaque-to-plaque distance, suggesting that desmoplakin is arranged with its long axis at an angle within the plaque. We next examined whether plaque organization changed in different adhesive states. Plaque-to-plaque distance for the desmoplakin rod and C-terminal domains decreased in PKP-1-mediated hyperadhesive desmosomes, suggesting that protein reorganization correlates with function. Finally, in human epidermis we found a difference in plaque-to-plaque distance for the desmoplakin C-terminal domain, but not the desmoplakin rod domain or plakoglobin, between basal and suprabasal cells. Our data reveal the molecular organization of desmosomes in cultured keratinocytes and skin as defined by dSTORM. PMID:27505428

  17. Insights on proximity effect and multiphoton induced luminescence from gold nanospheres in far field optical microscopy

    SciTech Connect

    Borglin, Johan; Guldbrand, Stina; Evenbratt, Hanne; Kirejev, Vladimir; Ericson, Marica B.; Grönbeck, Henrik

    2015-12-07

    Gold nanoparticles can be visualized in far-field multiphoton laser-scanning microscopy (MPM) based on the phenomena of multiphoton induced luminescence (MIL). This is of interest for biomedical applications, e.g., for cancer diagnostics, as MPM allows for working in the near-infrared (NIR) optical window of tissue. It is well known that the aggregation of particles causes a redshift of the plasmon resonance, but its implications for MIL applying far-field MPM should be further exploited. Here, we explore MIL from 10 nm gold nanospheres that are chemically deposited on glass substrates in controlled coverage gradients using MPM operating in NIR range. The substrates enable studies of MIL as a function of inter-particle distance and clustering. It was shown that MIL was only detected from areas on the substrates where the particle spacing was less than one particle diameter, or where the particles have aggregated. The results are interpreted in the context that the underlying physical phenomenon of MIL is a sequential two-photon absorption process, where the first event is driven by the plasmon resonance. It is evident that gold nanospheres in this size range have to be closely spaced or clustered to exhibit detectable MIL using far-field MPM operating in the NIR region.

  18. Multimodal imaging of lung tissue using optical coherence tomography and two photon microscopy

    NASA Astrophysics Data System (ADS)

    Gaertner, Maria; Cimalla, Peter; Geissler, Stefan; Meissner, Sven; Schnabel, Christian; Kuebler, Wolfgang M.; Koch, Edmund

    2012-02-01

    In the context of protective artificial ventilation strategies for patients with severe lung diseases, the contribution of ventilator settings to tissue changes on the alveolar level of the lung is still a question under debate. To understand the impact of respiratory settings as well as the dynamic process of respiration, high-resolution monitoring and visualization of the dynamics of lung alveoli are essential. An instrument allowing 3D imaging of lung tissue as well as imaging of functional constituents, such as elastin fibers, in in situ experimental conditions is presented in this study using a combination of Fourier domain optical coherence tomography (FD-OCT) and fiber-guided two photon microscopy. In a comparative study, fixed lung tissue, stained with sulforhodamine B for elastin fibers, was used to illustrate the ability of fiber-guided two photon excitation and single photon excitation for the visualization of elastin fibers within the tissue. Together with the fast 3D imaging capability of OCT, a new tool is given for the monitoring of alveolar lung dynamics in future in vivo experiments.

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

    NASA Astrophysics Data System (ADS)

    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.

  20. Quantitative morphological evaluation of laser ablation on calculus using full-field optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Xiao, Q.; Lü, T.; Li, Z.; Fu, L.

    2011-10-01

    The quantitative morphological evaluation at high resolution is of significance for the study of laser-tissue interaction. In this paper, a full-field optical coherence microscopy (OCM) system with high resolution of ˜2 μm was developed to investigate the ablation on urinary calculus by a free-running Er:YAG laser. We studied the morphological variation quantitatively corresponding to change of energy setting of the Er:YAG laser. The experimental results show that the full-field OCM enables quantitative evaluation of the morphological shape of craters and material removal, and particularly the fine structure. We also built a heat conduction model to simulate the process of laser-calculus interaction by using finite element method. Through the simulation, the removal region of the calculus was calculated according to the temperature distribution. As a result, the depth, width, volume, and the cross-sectional profile of the crater in calculus measured by full-field OCM matched well with the theoretical results based on the heat conduction model. Both experimental and theoretical results confirm that the thermal interaction is the dominant effect in the ablation of calculus by Er:YAG laser, demonstrating the effectiveness of full-field OCM in studying laser-tissue interactions.

  1. Cornea characterization using a combined multiphoton microscopy and optical coherence tomography system

    PubMed Central

    Lai, Tom; Tang, Shuo

    2014-01-01

    We present a multimodal imaging system which combines multiphoton microscopy and optical coherence tomography to visualize the morphological structures, and to quantify the refractive index (RI) and thickness of cornea. The morphological similarities and differences at different corneal layers across various species are identified. In the piscine and human corneas, the stromata exhibit thin fibers that indicate an overall collagen direction. Human corneas display collagen micro-folds which cause increased light attenuation. In the murine, porcine and bovine corneas, the stromata show interwoven collagen patterns. The Bowman’s layer and the Descemet’s membrane are also distinguished in some species. The RI and thicknesses are quantified for the epithelium and the stromal layers respectively, where the epithelium is found to have slightly higher RI than the stroma. The average epithelial and stromal RI are, respectively, 1.371 ± 0.016 and 1.360 ± 0.008 for the murine corneas; 1.502 ± 0.057 and 1.335 ± 0.011 for the piscine corneas; 1.433 ± 0.023 and 1.357 ± 0.013 for the human corneas; 1.476 ± 0.091 and 1.343 ± 0.013 for the porcine corneas; and 1.400 ± 0.007 and 1.376 ± 0.003 for the bovine corneas. The multimodal system can potentially provide a comprehensive characterization of the cornea. PMID:24877011

  2. Highly efficient plasmonic tip design for plasmon nanofocusing in near-field optical microscopy.

    PubMed

    Umakoshi, Takayuki; Saito, Yuika; Verma, Prabhat

    2016-03-14

    Near-field scanning optical microscopy (NSOM) combined with plasmon nanofocusing is a powerful nano-analytical tool due to its attractive feature of efficient background suppression as well as light energy compression to the nanoscale. In plasmon nanofocusing-based NSOM, the metallic tip plays an important role in inducing plasmon nanofocusing. It is, however, very challenging to control plasmonic properties of tips for plasmon nanofocusing with existing tip fabrication methods, even though the plasmonic properties need to be adjusted to experimental environments such as the sample or excitation wavelength. In this study, we propose an efficient tip design and fabrication which enable one to actively control plasmonic properties for efficient plasmon nanofocusing. Because our method offers flexibility in the material and structure of tips, one can easily modify the plasmonic properties depending on the requirements. Importantly, through optimization of the plasmonic properties, we achieve almost 100% reproducibility in plasmon nanofocusing in our experiments. This new approach of tip fabrication makes plasmon nanofocusing-based NSOM practical and reliable, and opens doors for many scientists working in related fields. PMID:26892672

  3. Quantitative Imaging of Rapidly Decaying Evanescent Fields Using Plasmonic Near-Field Scanning Optical Microscopy

    PubMed Central

    Zhang, Zhen; Ahn, Phillip; Dong, Biqin; Balogun, Oluwaseyi; Sun, Cheng

    2013-01-01

    Non-propagating evanescent fields play an important role in the development of nano-photonic devices. While detecting the evanescent fields in far-field can be accomplished by coupling it to the propagating waves, in practice they are measured in the presence of unwanted propagating background components. It leads to a poor signal-to-noise ratio and thus to errors in quantitative analysis of the local evanescent fields. Here we report on a plasmonic near-field scanning optical microscopy (p-NSOM) technique that incorporates a nanofocusing probe for adiabatic focusing of propagating surface plasmon polaritons at the probe apex, and for enhanced coupling of evanescent waves to the far-field. In addition, a harmonic demodulation technique is employed to suppress the contribution of the background. Our experimental results show strong evidence of background free near-field imaging using the new p-NSOM technique. Furthermore, we present measurements of surface plasmon cavity modes, and quantify their contributing sources using an analytical model. PMID:24076563

  4. Combining optical coherence tomography with fluorescence microscopy: a closer look into tissue

    NASA Astrophysics Data System (ADS)

    Gaertner, Maria; Cimalla, Peter; Knels, Lilla; Meissner, Sven; Koch, Edmund

    2010-04-01

    Optical coherence tomography (OCT) is a technique, capable of high resolution and non-invasive 3D imaging in vivo by detection of backscattered light from cellular and sub cellular structures. Due to visualization of micrometer sized tissue constituents and high penetration depths of up to 2 mm, it is already well established in medical fields like ophthalmology and dermatology. Laser scanning confocal microscopy (LSCM), on the contrary, gives further information on structural tissue components stained with suitable dyes. In combination, these two methods yield three dimensional and high resolution data providing geometrical and structural details of tissue. In this study, we present simultaneous OCT and LSCM image acquisition resulting in a lateral resolution of better than 6.2 μm for OCT and 0.8 μm for LSCM, respectively. The axial resolution of the OCT amounts to 8 μm. Two laser lines, 488 nm and 561 nm, are combined to provide fluorescence excitation of green and red dyes. By using a long working distance objective, it is possible to perform experiments on bulky specimens like isolated organs or animal models in vivo. First studies indicate the ability to identify strains of elastic fibers within lung tissue in combination with the three dimensional morphology of the lung.

  5. Xylem feeding by spittlebug nymphs: some observations by optical and cryo-scanning electron microscopy.

    PubMed

    Crews, L; McCully, M; Canny, M; Huang, C; Ling, L

    1998-04-01

    The feeding of spittlebug nymphs (Philaenus spumarius) from mature xylem vessels was studied by optical and cryo-analytical scanning electron microscopy. Feeding did not produce xylem embolisms and vessels remained liquid-filled during the day. Saliva secreted by the insect forms a hardened lining (salivary sheath) between the stylet bundle and the plant tissues. This sheath is continuous through the hole made by the stylets as they enter a vessel, and it extends into the vessel and along its periphery beyond the breach. The sheath is heterogeneous, with a thin outer layer adjoining the plant tissues and a thicker layer that contacts the stylet bundle. Both layers give positive histochemical reactions for proteins and, in fresh tissues, contain a red, strongly autofluorescent pigment, possibly condensed tannin derived from the plant (which is lost during tissue preparation), and other phenyl propanoid compounds, which are retained and which may produce the intense reaction of the periodic-acid-Schiff's-positive inner layer. It is concluded that the salivary sheath allows the insects to feed from functioning vessels without embolizing them or losing xylem fluid to the surrounding tissues. These findings and others in the entomological literature indicate low daytime tensions in the xylem conduits of the host plants. PMID:21684926

  6. Wide-field optical coherence microscopy of the mouse brain slice.

    PubMed

    Min, Eunjung; Lee, Junwon; Vavilin, Andrey; Jung, Sunwoo; Shin, Sungwon; Kim, Jeehyun; Jung, Woonggyu

    2015-10-01

    The imaging capability of optical coherence microscopy (OCM) has great potential to be used in neuroscience research because it is able to visualize anatomic features of brain tissue without labeling or external contrast agents. However, the field of view of OCM is still narrow, which dilutes the strength of OCM and limits its application. In this study, we present fully automated wide-field OCM for mosaic imaging of sliced mouse brains. A total of 308 segmented OCM images were acquired, stitched, and reconstructed as an en-face brain image after intensive imaging processing. The overall imaging area was 11.2×7.0  mm (horizontal×vertical), and the corresponding pixel resolution was 1.2×1.2  μm. OCM images were compared to traditional histology stained with Nissl and Luxol fast blue (LFB). In particular, the orientation of the fibers was analyzed and quantified in wide-field OCM. PMID:26421546

  7. Combining scanning haptic microscopy and fibre optic Raman spectroscopy for tissue characterization.

    PubMed

    Candefjord, S; Murayama, Y; Nyberg, M; Hallberg, J; Ramser, K; Ljungberg, B; Bergh, A; Lindahl, O A

    2012-08-01

    The tactile resonance method (TRM) and Raman spectroscopy (RS) are promising for tissue characterization in vivo. Our goal is to combine these techniques into one instrument, to use TRM for swift scanning, and RS for increasing the diagnostic power. The aim of this study was to determine the classification accuracy, using support vector machines, for measurements on porcine tissue and also produce preliminary data on human prostate tissue. This was done by developing a new experimental set-up combining micro-scale TRM-scanning haptic microscopy (SHM)-for assessing stiffness on a micro-scale, with fibre optic RS measurements for assessing biochemical content. We compared the accuracy using SHM alone versus SHM combined with RS, for different degrees of tissue homogeneity. The cross-validation classification accuracy for healthy porcine tissue types using SHM alone was 65-81%, and when RS was added it increased to 81-87%. The accuracy for healthy and cancerous human tissue was 67-70% when only SHM was used, and increased to 72-77% for the combined measurements. This shows that the potential for swift and accurate classification of healthy and cancerous prostate tissue is high. This is promising for developing a tool for probing the surgical margins during prostate cancer surgery. PMID:22762445

  8. Multimodal nonlinear optical microscopy improves the accuracy of early diagnosis of squamous intraepithelial neoplasia

    NASA Astrophysics Data System (ADS)

    Teh, Seng Khoon; Zheng, Wei; Li, Shuxia; Li, Dong; Zeng, Yan; Yang, Yanqi; Qu, Jianan Y.

    2013-03-01

    We explore diagnostic utility of a multicolor excitation multimodal nonlinear optical (NLO) microscopy for noninvasive detection of squamous epithelial precancer in vivo. The 7,12-dimenthylbenz(a)anthracene treated hamster cheek pouch was used as an animal model of carcinogenesis. The NLO microscope system employed was equipped with the ability to collect multiple tissue endogenous NLO signals such as two-photon excited fluorescence of keratin, nicotinamide adenine dinucleotide, collagen, and tryptophan, and second harmonic generation of collagen in spectral and time domains simultaneously. A total of 34 (11 controlled and 23 treated) Golden Syrian hamsters with 62 in vivo spatially distinct measurement sites were assessed in this study. High-resolution label-free NLO images were acquired from stratum corneum, stratum granulosum-stratum basale, and stroma for all tissue measurement sites. A total of nine and eight features from 745 and 600 nm excitation wavelengths, respectively, involving tissue structural and intrinsic biochemical properties were found to contain significant diagnostic information for precancers detection (p<0.05). Particularly, 600 nm excited tryptophan fluorescence signals emanating from stratum corneum was revealed to provide remarkable diagnostic utility. Multivariate statistical techniques confirmed the integration of diagnostically significant features from multicolor excitation wavelengths yielded improved diagnostic accuracy as compared to using the individual wavelength alone.

  9. Real Time Gabor-Domain Optical Coherence Microscopy for 3D Imaging.

    PubMed

    Rolland, Jannick P; Canavesi, Cristina; Tankam, Patrice; Cogliati, Andrea; Lanis, Mara; Santhanam, Anand P

    2016-01-01

    Fast, robust, nondestructive 3D imaging is needed for the characterization of microscopic tissue structures across various clinical applications. A custom microelectromechanical system (MEMS)-based 2D scanner was developed to achieve, together with a multi-level GPU architecture, 55 kHz fast-axis A-scan acquisition in a Gabor-domain optical coherence microscopy (GD-OCM) custom instrument. GD-OCM yields high-definition micrometer-class volumetric images. A dynamic depth of focusing capability through a bio-inspired liquid lens-based microscope design, as in whales' eyes, was developed to enable the high definition instrument throughout a large field of view of 1 mm3 volume of imaging. Developing this technology is prime to enable integration within the workflow of clinical environments. Imaging at an invariant resolution of 2 μm has been achieved throughout a volume of 1 × 1 × 0.6 mm3, acquired in less than 2 minutes. Volumetric scans of human skin in vivo and an excised human cornea are presented. PMID:27046601

  10. Closing the gap: The approach of optical and computational microscopy to uncover biomembrane organization.

    PubMed

    Eggeling, Christian; Honigmann, Alf

    2016-10-01

    Biological membranes are complex composites of lipids, proteins and sugars, which catalyze a myriad of vital cellular reactions in a spatiotemporal tightly controlled manner. Our understanding of the organization principles of biomembranes is limited mainly by the challenge to measure distributions and interactions of lipids and proteins within the complex environment of living cells. With the recent advent of super-resolution optical microscopy (or nanoscopy) one now has approached the molecular scale regime with non-invasive live cell fluorescence observation techniques. Since in silico molecular dynamics (MD) simulation techniques are also improving to study larger and more complex systems we can now start to integrate live-cell and in silico experiments to develop a deeper understanding of biomembranes. In this review we summarize recent progress to measure lipid-protein interactions in living cells and give examples how MD simulations can complement and upgrade the experimental data. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:27039279

  11. Dual tree complex wavelet transform based denoising of optical microscopy images.

    PubMed

    Bal, Ufuk

    2012-12-01

    Photon shot noise is the main noise source of optical microscopy images and can be modeled by a Poisson process. Several discrete wavelet transform based methods have been proposed in the literature for denoising images corrupted by Poisson noise. However, the discrete wavelet transform (DWT) has disadvantages such as shift variance, aliasing, and lack of directional selectivity. To overcome these problems, a dual tree complex wavelet transform is used in our proposed denoising algorithm. Our denoising algorithm is based on the assumption that for the Poisson noise case threshold values for wavelet coefficients can be estimated from the approximation coefficients. Our proposed method was compared with one of the state of the art denoising algorithms. Better results were obtained by using the proposed algorithm in terms of image quality metrics. Furthermore, the contrast enhancement effect of the proposed method on collagen fıber images is examined. Our method allows fast and efficient enhancement of images obtained under low light intensity conditions. PMID:23243573

  12. Resolution enhancement of digital laser scanning fluorescence microscopy with a dual-lens optical pickup head

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

    The resolution of the cell fluorescence image captured by a digital laser scanning microscopy with a modified dual-lens BD-ROM optical pickup head is enhanced by image registration and double sample frequency. A dual objective lens of red (655 nm) and blue (405 or 488 nm) laser sources with numerical apertures of 0.6 and 0.85 is used for sample focusing and position tracking and cell fluorescence image capturing, respectively. The image registration and capturing frequency are based on the address-coded patterns of a sample slide. The address-coded patterns are designed as a string of binary code, which comprises a plurality of base-straight lands and grooves and data-straight grooves. The widths of the base-straight lands, base-straight grooves, and data-straight grooves are 0.38, 0.38, and 0.76 μm, respectively. The numbers of sample signals in the x-direction are measured at every intersection point by intersecting the base intensity of the push-pull signal of the address-coded patterns, which has a minimum spacing of 0.38 μm. After taking a double sample frequency, the resolution of the measured cell fluorescence image is enhanced from 0.38 μm to the diffraction limit of the objective lens.

  13. Chloride-dependent secretion of alveolar wall liquid determined by optical-sectioning microscopy.

    PubMed

    Lindert, Jens; Perlman, Carrie E; Parthasarathi, Kaushik; Bhattacharya, Jahar

    2007-06-01

    The liquid layer lining the pulmonary alveolar wall critically determines the lung's immune defense against inhaled pathogens, because it provides a liquid milieu in the air-filled alveolus for dispersal of immune cells and defensive surfactant proteins. However, mechanisms underlying formation of the liquid are unknown. We achieved visualization of the alveolar wall liquid (AWL) in situ in mouse lungs by means of optical-sectioning microscopy. Continuous liquid secretion was present in alveoli of wild-type (WT) mice under baseline conditions. This secretion was blocked by inhibitors of the cystic fibrosis transmembrane regulator (CFTR). The secretion was absent in Cftr(-/-) mice, and it was blocked when chloride was depleted from the perfusate of WT mice, providing the first evidence that CFTR-dependent chloride secretion causes AWL formation. Injected microparticles demonstrated flow of the AWL. The flow was blocked by CFTR inhibition and was absent in Cftr(-/-) mice. We conclude that CFTR-dependent liquid secretion is present in alveoli of the adult mouse. Defective alveolar secretion might impair alveolar immune defense and promote alveolar disease. PMID:17290033

  14. Combined two-photon microscopy and optical coherence tomography using individually optimized sources

    NASA Astrophysics Data System (ADS)

    Jeong, Bosu; Lee, Byunghak; Jang, Min Seong; Nam, Hyoseok; Kim, Hae Koo; Yoon, Sang June; Doh, Junsang; Lee, Sang-Joon; Yang, Bo-Gie; Jang, Myoung Ho; Kim, Ki Hean

    2011-03-01

    Two-photon microscopy (TPM) and optical coherence tomography (OCT) are 3D tissue imaging techniques based on different contrast mechanisms. We developed a combined system of TPM and OCT to provide information of both imaging modalities for in-vivo tissue study. TPM and OCT were implemented by using separate light sources, a Ti-Sapphire laser and a wavelength-swept source centered at 1300 nm respectively, and scanners. Light from the two sources was combined for the simultaneous imaging of tissue samples. TPM provided molecular, cellular information of tissues in the region of a few hundred microns on one side at a sub-cellular resolution, and ran at approximately 40 frames per second. OCT provided structural information in the tissue region larger than TPM images at a sub-tenth micron resolution by using 0.1 numerical aperture. OCT had the field of view of 800 um × 800 um based on a 20x objective, the sensitivity of 97dB, and the imaging speed of 0.8 volumes per second. This combined system was tested with simple microsphere specimens, and then was applied to image the explanted intestine of a mouse model and the plant leaves. Morphology and micro-structures of the intestine villi and immune cells within the villi were shown in the intestine image, and chloroplasts and various microstructures of the maize leaves were visualized in 3D by the combined system.

  15. Determining the equation of state of amorphous solids at high pressure using optical microscopy

    NASA Astrophysics Data System (ADS)

    Amin, Samrat A.; Rissi, Erin N.; McKiernan, Keri; Yarger, Jeffery L.

    2012-03-01

    A method to determine the volumetric equation of state of amorphous solids using optical microscopy in a diamond anvil cell is described. The method relies on two- dimensional image acquisition and analysis to quantify changes in the projected image area with compression. The area analysis methods prove to be robust against improper focusing and lighting conditions while providing the accuracy necessary to deduce small area changes, which correspond to small volume changes in an isotropic material. The image capture, area analysis method is used to determine the compression of cubic crystals, yielding results in good agreement with diffraction and volumetric measurements. As a proof of concept, the equation of state of amorphous red phosphorus is measured up to 12 GPa under hydrostatic conditions where the quantified volume change between the red and black phases is approximately ΔV/V0 ≈ 0.05 after a reduction of volume nearing 35% at 8 GPa. A large hysteresis is present during decompression and eventually results in a 15% permanent densification relative to the starting volume, which is attributed to a pressure induced crystallization from red to black phosphorus. The robustness of the technique is also demonstrated by measuring the equation of state of GeSe2 glasses for semi transparent samples and As2O3 in which gold coating is used as a contrasting aid.

  16. Highly efficient plasmonic tip design for plasmon nanofocusing in near-field optical microscopy

    NASA Astrophysics Data System (ADS)

    Umakoshi, Takayuki; Saito, Yuika; Verma, Prabhat

    2016-03-01

    Near-field scanning optical microscopy (NSOM) combined with plasmon nanofocusing is a powerful nano-analytical tool due to its attractive feature of efficient background suppression as well as light energy compression to the nanoscale. In plasmon nanofocusing-based NSOM, the metallic tip plays an important role in inducing plasmon nanofocusing. It is, however, very challenging to control plasmonic properties of tips for plasmon nanofocusing with existing tip fabrication methods, even though the plasmonic properties need to be adjusted to experimental environments such as the sample or excitation wavelength. In this study, we propose an efficient tip design and fabrication which enable one to actively control plasmonic properties for efficient plasmon nanofocusing. Because our method offers flexibility in the material and structure of tips, one can easily modify the plasmonic properties depending on the requirements. Importantly, through optimization of the plasmonic properties, we achieve almost 100% reproducibility in plasmon nanofocusing in our experiments. This new approach of tip fabrication makes plasmon nanofocusing-based NSOM practical and reliable, and opens doors for many scientists working in related fields.

  17. Mapping fluxes of radicals from the combination of electrochemical activation and optical microscopy.

    PubMed

    Munteanu, Sorin; Roger, Jean Paul; Fedala, Yasmina; Amiot, Fabien; Combellas, Catherine; Tessier, Gilles; Kanoufi, Frédéric

    2013-01-01

    The coating of gold (Au) electrode surfaces with nitrophenyl (NP) layers is studied by combination of electrochemical actuation and optical detection. The electrochemical actuation of the reduction of the nitrobenzenediazonium (NBD) precursor is used to generate NP radicals and therefore initiate the electrografting. The electrografting process is followed in situ and in real time by light reflectivity microscopy imaging, allowing for spatio-temporal imaging with sub-micrometer lateral resolution and sub-nanometer thickness sensitivity of the local growth of a transparent organic coating onto a reflecting Au electrode. The interest of the electrochemical actuation resides in its ability to finely control the grafting rate of the NP layer through the electrode potential. Coupling the electrochemical actuation with microscopic imaging of the electrode surface allows quantitative estimates of the local grafting rates and subsequently a real time and in situ mapping of the reacting fluxes of NP radicals on the surface. Over the 2 orders of magnitude range of grafting rates (from 0.04 to 4 nm s(-1)), it is demonstrated that the edge of Au electrodes are grafted -1.3 times more quickly than their centre, illustrating the manifestation of edge-effects on flux distribution at an electrode. A model is proposed to explain the observed edge-effect, it relies on the short lifetime of the intermediate NP radical species. PMID:24466667

  18. Towards optically-integrated scanning tunneling microscopy studies of defects in semiconductors

    NASA Astrophysics Data System (ADS)

    Benjamin, Anne; Lang, Evan; Werner, Kevin; Chowdhury, Enam; Gupta, Jay

    As electronic devices approach the nanoscale, their function is increasingly dependent on the local environment of individual defects. We are developing a combination of optical illumination and scanning tunneling microscopy techniques to study how the properties of individual defects depend on aspects of the local environment, such surface or defect proximity, applied electric fields, and illumination. Here we present studies of individual Zn and Er impurities in GaAs(110).We use controlled motion of the STM tip during voltage sweeps to resolve previously hidden in-gap states of Zn acceptors and probe Zn further from the surface than previously accessible. We discovered two classes of Zn acceptors, one with defect states that did not shift with tip-induced band bending (TIBB), and one with states that do. Similar behavior was observed for above-gap illumination, consistent with the surface photovoltage effect (SPV). For Er on GaAs(110), we discovered three different adsorption states sharing two different sites. We found defect states near the conduction band edge, which shifted with TIBB as well as IR illumination resonant with the Er f-shell transitions.

  19. Simultaneous confocal fluorescence microscopy and optical coherence tomography for drug distribution and tissue integrity assessment

    NASA Astrophysics Data System (ADS)

    Rinehart, Matthew T.; LaCroix, Jeffrey; Henderson, Marcus; Katz, David; Wax, Adam

    2011-03-01

    The effectiveness of microbicidal gels, topical products developed to prevent infection by sexually transmitted diseases including HIV/AIDS, is governed by extent of gel coverage, pharmacokinetics of active pharmaceutical ingredients (APIs), and integrity of vaginal epithelium. While biopsies provide localized information about drug delivery and tissue structure, in vivo measurements are preferable in providing objective data on API and gel coating distribution as well as tissue integrity. We are developing a system combining confocal fluorescence microscopy with optical coherence tomography (OCT) to simultaneously measure local concentrations and diffusion coefficients of APIs during transport from microbicidal gels into tissue, while assessing tissue integrity. The confocal module acquires 2-D images of fluorescent APIs multiple times per second allowing analysis of lateral diffusion kinetics. The custom Fourier domain OCT module has a maximum a-scan rate of 54 kHz and provides depth-resolved tissue integrity information coregistered with the confocal fluorescence measurements. The combined system is validated by imaging phantoms with a surrogate fluorophore. Time-resolved API concentration measured at fixed depths is analyzed for diffusion kinetics. This multimodal system will eventually be implemented in vivo for objective evaluation of microbicide product performance.

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

    PubMed Central

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

    2010-01-01

    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. PMID:21054095

  1. Nonlinear optical response of the collagen triple helix and second harmonic microscopy of collagen liquid crystals

    NASA Astrophysics Data System (ADS)

    Deniset-Besseau, A.; De Sa Peixoto, P.; Duboisset, J.; Loison, C.; Hache, F.; Benichou, E.; Brevet, P.-F.; Mosser, G.; Schanne-Klein, M.-C.

    2010-02-01

    Collagen is characterized by triple helical domains and plays a central role in the formation of fibrillar and microfibrillar networks, basement membranes, as well as other structures of the connective tissue. Remarkably, fibrillar collagen exhibits efficient Second Harmonic Generation (SHG) and SHG microscopy proved to be a sensitive tool to score fibrotic pathologies. However, the nonlinear optical response of fibrillar collagen is not fully characterized yet and quantitative data are required to further process SHG images. We therefore performed Hyper-Rayleigh Scattering (HRS) experiments and measured a second order hyperpolarisability of 1.25 10-27 esu for rat-tail type I collagen. This value is surprisingly large considering that collagen presents no strong harmonophore in its amino-acid sequence. In order to get insight into the physical origin of this nonlinear process, we performed HRS measurements after denaturation of the collagen triple helix and for a collagen-like short model peptide [(Pro-Pro-Gly)10]3. It showed that the collagen large nonlinear response originates in the tight alignment of a large number of weakly efficient harmonophores, presumably the peptide bonds, resulting in a coherent amplification of the nonlinear signal along the triple helix. To illustrate this mechanism, we successfully recorded SHG images in collagen liquid solutions by achieving liquid crystalline ordering of the collagen triple helices.

  2. Line-scan Raman microscopy complements optical coherence tomography for tumor boundary detection

    NASA Astrophysics Data System (ADS)

    Sudheendran, Narendran; Qi, Ji; Young, Eric D.; Lazar, Alexander J.; Lev, Dina C.; Pollock, Raphael E.; Larin, Kirill V.; Shih, Wei-Chuan

    2014-10-01

    Current technique for tumor resection requires biopsy of the tumor region and histological confirmation before the surgeon can be certain that the entire tumor has been resected. This confirmation process is time consuming both for the surgeon and the patient and also requires sacrifice of healthy tissue, motivating the development of novel technologies which can enable real-time detection of tumor-healthy tissue boundary for faster and more efficient surgeries. In this study, the potential of combining structural information from optical coherence tomography (OCT) and molecular information from line-scan Raman microscopy (LSRM) for such an application is presented. The results show a clear presence of boundary between myxoid liposarcoma and normal fat which is easily identifiable both from structural and molecular information. In cases where structural images are indistinguishable, for example, in normal fat and well differentiated liposarcoma (WDLS) or gastrointestinal sarcoma tumor (GIST) and myxoma, distinct molecular spectra have been obtained. The results suggest LSRM can effectively complement OCT to tumor boundary demarcation with high specificity.

  3. Confocal scanning optical microscopy of a 3-million-year-old Australopithecus afarensis femur.

    PubMed

    Bromage, T G; Goldman, H M; McFarlin, S C; Perez Ochoa, A; Boyde, A

    2009-01-01

    Portable confocal scanning optical microscopy (PCSOM) has been specifically developed for the noncontact and nondestructive imaging of early human fossil hard tissues, which here we describe and apply to a 3-million-year-old femur from the celebrated Ethiopian skeleton, "Lucy," referred to Australopithecus afarensis. We examine two bone tissue parameters that demonstrate the potential of this technology. First, subsurface reflection images from intact bone reveal bone cell spaces, the osteocyte lacunae, whose density is demonstrated to scale negatively with body size, reflecting aspects of metabolism and organismal life history. Second, images of a naturally fractured cross section near to Lucy's femoral mid-shaft, which match in sign those of transmitted circularly polarized light, reveal relative collagen fiber orientation patterns that are an important indicator of femoral biomechanical efficacy. Preliminary results indicate that Lucy was characterized by metabolic constraints typical for a primate her body size and that in her femur she was adapted to habitual bipedalism. Limitations imposed by the transport and invasive histology of unique or rare fossils motivated development of the PCSOM so that specimens may be examined wherever and whenever nondestructive imaging is required. PMID:19191265

  4. Imaging dynamic processes using fiber laser optical-resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Shi, Wei; Shao, Peng; Hajireza, Parsin; Forbrich, Alexander; Zemp, Roger J.

    2012-02-01

    Recently we have reported in vivo near-real-time volumetric optical-resolution photoacoustic microscopy (OR-PAM) using a high pulse-repetition-rate (PRR) nanosecond fiber-laser to realize 2 volumetric image frames per second (fps) within 1mm × 1mm field of view (FOV). Based on our previous OR-PAM system, we are developing a label-free realtime OR-PAM system in reflection mode for higher frame-rates. The system permits imaging of microcirculation hemodynamics, and helps make the technology easier to use for biologists, providing real-time feedback for focusing and positioning. Using a nanosecond-pulsed 532-nm fiber laser combined with fast-scanning mirrors, our proposed system demonstrated its capability of sustained in vivo imaging of horizontal and vertical translation at 0.5 fps for 1mm × 1mm FOV (400 × 400 pixels). Also, real-time in vivo imaging of blood flow at 30 fps for 250μm × 250μm FOV (100 × 100 pixels) was demonstrated. It is anticipated that the real-time nature of the system should prove important in clinical and preclinical adaption, and may prove useful for functional brain imaging studies.

  5. Advanced magneto-optical microscopy: Imaging from picoseconds to centimeters - imaging spin waves and temperature distributions (invited)

    NASA Astrophysics Data System (ADS)

    Urs, Necdet Onur; Mozooni, Babak; Mazalski, Piotr; Kustov, Mikhail; Hayes, Patrick; Deldar, Shayan; Quandt, Eckhard; McCord, Jeffrey

    2016-05-01

    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.

  6. Local optical absorption spectra of h-BN–MoS2 van der Waals heterostructure revealed by scanning near-field optical microscopy

    NASA Astrophysics Data System (ADS)

    Nozaki, Junji; Kobayashi, Yu; Miyata, Yasumitsu; Maniwa, Yutaka; Watanabe, Kenji; Taniguchi, Takashi; Yanagi, Kazuhiro

    2016-06-01

    Van der Waals (vdW) heterostructures, in which different two-dimensional layered materials are stacked, can exhibit unprecedented optical properties. Development of a technique to clarify local optical properties of vdW heterostructures is of great importance for the correct understanding of their backgrounds. Here, we examined local optical absorption spectra of h-BN–MoS2 vdW heterostructures by scanning near-field microscopy measurements with a spatial resolution of 100 nm. In an as-grown sample, there was almost no site dependence of their optical absorption spectra. However, in a degraded sample where defects and deformations were artificially induced, a significant site-dependence of optical absorption spectra was observed.

  7. Asymmetric-detection time-stretch optical microscopy (ATOM) for ultrafast high-contrast cellular imaging in flow

    PubMed Central

    Wong, Terence T. W.; Lau, Andy K. S.; Ho, Kenneth K. Y.; Tang, Matthew Y. H.; Robles, Joseph D. F.; Wei, Xiaoming; Chan, Antony C. S.; Tang, Anson H. L.; Lam, Edmund Y.; Wong, Kenneth K. Y.; Chan, Godfrey C. F.; Shum, Ho Cheung; Tsia, Kevin K.

    2014-01-01

    Accelerating imaging speed in optical microscopy is often realized at the expense of image contrast, image resolution, and detection sensitivity – a common predicament for advancing high-speed and high-throughput cellular imaging. We here demonstrate a new imaging approach, called asymmetric-detection time-stretch optical microscopy (ATOM), which can deliver ultrafast label-free high-contrast flow imaging with well delineated cellular morphological resolution and in-line optical image amplification to overcome the compromised imaging sensitivity at high speed. We show that ATOM can separately reveal the enhanced phase-gradient and absorption contrast in microfluidic live-cell imaging at a flow speed as high as ~10 m/s, corresponding to an imaging throughput of ~100,000 cells/sec. ATOM could thus be the enabling platform to meet the pressing need for intercalating optical microscopy in cellular assay, e.g. imaging flow cytometry – permitting high-throughput access to the morphological information of the individual cells simultaneously with a multitude of parameters obtained in the standard assay. PMID:24413677

  8. Analytic 3D Imaging of Mammalian Nucleus at Nanoscale Using Coherent X-Rays and Optical Fluorescence Microscopy

    PubMed Central

    Song, Changyong; Takagi, Masatoshi; Park, Jaehyun; Xu, Rui; Gallagher-Jones, Marcus; Imamoto, Naoko; Ishikawa, Tetsuya

    2014-01-01

    Despite the notable progress that has been made with nano-bio imaging probes, quantitative nanoscale imaging of multistructured specimens such as mammalian cells remains challenging due to their inherent structural complexity. Here, we successfully performed three-dimensional (3D) imaging of mammalian nuclei by combining coherent x-ray diffraction microscopy, explicitly visualizing nuclear substructures at several tens of nanometer resolution, and optical fluorescence microscopy, cross confirming the substructures with immunostaining. This demonstrates the successful application of coherent x-rays to obtain the 3D ultrastructure of mammalian nuclei and establishes a solid route to nanoscale imaging of complex specimens. PMID:25185543

  9. Bases for time-resolved probing of transient carrier dynamics by optical pump-probe scanning tunneling microscopy

    NASA Astrophysics Data System (ADS)

    Yokota, Munenori; Yoshida, Shoji; Mera, Yutaka; Takeuchi, Osamu; Oigawa, Haruhiro; Shigekawa, Hidemi

    2013-09-01

    The tangled mechanism that produces optical pump-probe scanning tunneling microscopy spectra from semiconductors was analyzed by comparing model simulation data with experimental data. The nonlinearities reflected in the spectra, namely, the excitations generated by paired laser pulses with a delay time, the logarithmic relationship between carrier density and surface photovoltage (SPV), and the effect of the change in tunneling barrier height depending on SPV, were examined along with the delay-time-dependent integration process used in measurement. The optimum conditions required to realize reliable measurement, as well as the validity of the microscopy technique, were demonstrated for the first time.

  10. Optical and tunneling microscopy and spectroscopy at the ultimate spatial limit

    NASA Astrophysics Data System (ADS)

    Chen, Chi

    2009-12-01

    The combination of optical detection system with a scanning tunneling microscope (STM) leads to the possibility of resolving radiative transition probability with the ultrahigh spatial resolution of STM in real space. This opens an innovative approach toward revealing the correlation between molecular structure, electronic characteristics, and optical properties. This thesis describes a series of experiments that manifests this correlation, including atomic silver chains and single porphine molecules. In atomic silver chains, the number and positions of the emission maxima in the photon images match the nodes in the dI/d V images of "particle-in-a-box" states. This surprising correlation between the emission maxima and nodes in the density of states is a manifestation of Fermi's golden rule in real space for radiative transitions, which provides an understanding of the mechanism of STM induced light emission. From single porphine molecules, orthogonal spatial contrast of two types of vibronic coupling is resolved by both photon spectroscopy and vibronic-mode-selected photon images. Intramolecular transitions from the two orthogonal LUMOs individually couple to different molecular normal modes. This is the first demonstration of the photon emission probability of a single molecule and its direct correlations with the molecular orbitals. This also provides the first real space experimental evidence to separate the tangled effects of molecular conformations and nano-environments on the inhomogeneity of molecular emission. DSB molecules are found to have two conformational isomers and one of them shows surface chirality. All these conformers and enantiomers can be switched to each other by electron injection. Different DSB conformers present distinct manipulation dynamics, which demonstrate how different conformations and their preferred adsorption geometries can have pronounced influence on the molecular mechanics on the surface. Overall, this thesis studies the very

  11. Advances in combined endoscopic fluorescence confocal microscopy and optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Risi, Matthew D.

    Confocal microendoscopy provides real-time high resolution cellular level images via a minimally invasive procedure. Results from an ongoing clinical study to detect ovarian cancer with a novel confocal fluorescent microendoscope are presented. As an imaging modality, confocal fluorescence microendoscopy typically requires exogenous fluorophores, has a relatively limited penetration depth (100 μm), and often employs specialized aperture configurations to achieve real-time imaging in vivo. Two primary research directions designed to overcome these limitations and improve diagnostic capability are presented. Ideal confocal imaging performance is obtained with a scanning point illumination and confocal aperture, but this approach is often unsuitable for real-time, in vivo biomedical imaging. By scanning a slit aperture in one direction, image acquisition speeds are greatly increased, but at the cost of a reduction in image quality. The design, implementation, and experimental verification of a custom multi-point-scanning modification to a slit-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution while maintaining real-time imaging rates. In addition, the multi-point aperture geometry greatly reduces the effects of tissue scatter on imaging performance. Optical coherence tomography (OCT) has seen wide acceptance and FDA approval as a technique for ophthalmic retinal imaging, and has been adapted for endoscopic use. As a minimally invasive imaging technique, it provides morphological characteristics of tissues at a cellular level without requiring the use of exogenous fluorophores. OCT is capable of imaging deeper into biological tissue (˜1-2 mm) than confocal fluorescence microscopy. A theoretical analysis of the use of a fiber-bundle in spectral-domain OCT systems is presented. The fiber-bundle enables a flexible endoscopic design and provides fast, parallelized acquisition of the optical coherence tomography

  12. Computational optical-sectioning microscopy for 3D quantization of cell motion: results and challenges

    NASA Astrophysics Data System (ADS)

    McNally, James G.

    1994-09-01

    How cells move and navigate within a 3D tissue mass is of central importance in such diverse problems as embryonic development, wound healing and metastasis. This locomotion can now be visualized and quantified by using computation optical-sectioning microscopy. In this approach, a series of 2D images at different depths in a specimen are stacked to construct a 3D image, and then with a knowledge of the microscope's point-spread function, the actual distribution of fluorescent intensity in the specimen is estimated via computation. When coupled with wide-field optics and a cooled CCD camera, this approach permits non-destructive 3D imaging of living specimens over long time periods. With these techniques, we have observed a complex diversity of motile behaviors in a model embryonic system, the cellular slime mold Dictyostelium. To understand the mechanisms which control these various behaviors, we are examining motion in various Dictyostelium mutants with known defects in proteins thought to be essential for signal reception, cell-cell adhesion or locomotion. This application of computational techniques to analyze 3D cell locomotion raises several technical challenges. Image restoration techniques must be fast enough to process numerous 1 Gbyte time-lapse data sets (16 Mbytes per 3D image X 60 time points). Because some cells are weakly labeled and background intensity is often high due to unincorporated dye, the SNR in some of these images is poor. Currently, the images are processed by a regularized linear least- squares restoration method, and occasionally by a maximum-likelihood method. Also required for these studies are accurate automated- tracking procedures to generate both 3D trajectories for individual cells and 3D flows for a group of cells. Tracking is currently done independently for each cell, using a cell's image as a template to search for a similar image at the next time point. Finally, sophisticated visualization techniques are needed to view the

  13. In-situ imaging of reacting single-particle zeolites by non-linear optical microscopy

    NASA Astrophysics Data System (ADS)

    Wrzesinski, Paul J.; Slipchenko, Mikhail N.; Zaman, Taslima A.; Rioux, Robert M.; Gord, James R.; Roy, Sukesh

    2015-03-01

    Zeolite catalysis has been exploited by the petrochemical industry since the 1940's for catalytic cracking reactions of long chain hydrocarbons. The selectivity of zeolites strongly depends on a pore size, which is controlled by the chosen structure-directing agent (SDA) and by the SDA decomposition/removal process. Although zeolites are composed of micron-sized crystals, studies of zeolite materials typically focus on bulk (i.e., ensemble) measurements to elucidate structure-function information or to optimize catalysts and/or process parameters. To examine these phenomena on the microscale, non-linear optical microscopy is used to provide real-time imaging of chemical reactions in zeolites at temperatures exceeding 400°C. The template decomposition mechanism is studied, as elucidation of the mechanism is critical to understanding the relationship between the decomposition chemistry and the nanoscale features of the zeolite (topology, Si/Al ratio, added dopants). Forward stimulated Raman scattering (SRS), forward coherent anti-Stokes Raman scattering (CARS) and epi two-photon fluorescence (TPF) modalities are acquired simultaneously providing video-rate structural and chemical information. A high-temperature cell with gas inlet system is used for the study of reactions under various temperatures and gas environments. Examining the decomposition process with single-particle resolution enables access to ensemble-level and spatially-resolved behavior. Parallel experiments on bulk zeolite powders are conducted to enable comparison of ensemble and single-particle behavior during template decomposition. Our multi-technique approach has high potential for gaining insight into the link between nanoscale structure and catalytic activity and selectivity of zeolitic materials.

  14. Quantitative Assessment of UVA-Riboflavin Corneal Cross-Linking Using Nonlinear Optical Microscopy

    PubMed Central

    Chai, Dongyul; Gaster, Ronald N.; Roizenblatt, Roberto; Juhasz, Tibor; Brown, Donald J.

    2011-01-01

    Purpose. Corneal collagen cross-linking (CXL) by the use of riboflavin and ultraviolet-A light (UVA) is a promising and novel treatment for keratoconus and other ectatic disorders. Since CXL results in enhanced corneal stiffness, this study tested the hypothesis that CXL-induced stiffening would be proportional to the collagen autofluorescence intensity measured with nonlinear optical (NLO) microscopy. Methods. Rabbit eyes (n = 50) were separated into five groups including: (1) epithelium intact; (2) epithelium removed; (3) epithelium removed and soaked in riboflavin, (4) epithelium removed and soaked in riboflavin, with 15 minutes of UVA exposure; and (5) epithelium removed and soaked in riboflavin, with 30 minutes of UVA exposure. Corneal stiffness was quantified by measuring the force required to displace the cornea 500 μm. Corneas were then fixed in paraformaldehyde and sectioned, and the collagen autofluorescence over the 400- to 450-nm spectrum was recorded. Results. There was no significant difference in corneal stiffness among the three control groups. Corneal stiffness was significantly and dose dependently increased after UVA (P < 0.0005). Autofluorescence was detected only within the anterior stroma of the UVA-treated groups, with no significant difference in the depth of autofluorescence between different UVA exposure levels. The signal intensity was also significantly increased with longer UVA exposure (P < 0.001). Comparing corneal stiffness with autofluorescence intensity revealed a significant correlation between these values (R2 = 0.654; P < 0.0001). Conclusions. The results of this study indicate a significant correlation between corneal stiffening and the intensity of collagen autofluorescence after CXL. This finding suggests that the efficacy of CXL in patients could be monitored by assessing collagen autofluorescence. PMID:21508101

  15. Ray-tracing study on the post-scanner variable beam expansion optics in a two-photon microscopy system

    NASA Astrophysics Data System (ADS)

    Kim, Do-Hyun; Welle, Cristin; Krauthamer, Victor

    2012-03-01

    Due to the low signal levels typical of two-photon microscopy (TPM) in biological samples, optical design optimization is critical. One of the most important factors is overfilling of the back aperture of the objective lens. A variable beam expander is commonly placed before the scanning mirrors to achieve this goal, however, this may cause degradation of image quality due to increased dispersion. Additionally, scanning mirror size restricts the degree of expansion, which often prevents the overfilling of objective lens back aperture. We investigated the implementation of variable beam expansion optics after the scanning mirrors. Ray-tracing analyses confirmed that the post-scanner beam expansion has two key advantages over the conventional pre-scanner beam expansion approach: decreasing the number of optical elements reduces pulse dispersion and reducing the size of the scanning mirror enables faster scanning. Resolution and aberration of a TPM with post-scanner beam expansion optics were analysed.

  16. All-optical regulation of gene expression in targeted cells

    NASA Astrophysics Data System (ADS)

    Wang, Yisen; He, Hao; Li, Shiyang; Liu, Dayong; Lan, Bei; Hu, Minglie; Cao, Youjia; Wang, Chingyue

    2014-06-01

    Controllable gene expression is always a challenge and of great significance to biomedical research and clinical applications. Recently, various approaches based on extra-engineered light-sensitive proteins have been developed to provide optogenetic actuators for gene expression. Complicated biomedical techniques including exogenous genes engineering, transfection, and material delivery are needed. Here we present an all-optical method to regulate gene expression in targeted cells. Intrinsic or exogenous genes can be activated by a Ca2+-sensitive transcription factor nuclear factor of activated T cells (NFAT) driven by a short flash of femtosecond-laser irradiation. When applied to mesenchymal stem cells, expression of a differentiation regulator Osterix can be activated by this method to potentially induce differentiation of them. A laser-induced ``Ca2+-comb'' (LiCCo) by multi-time laser exposure is further developed to enhance gene expression efficiency. This noninvasive method hence provides an encouraging advance of gene expression regulation, with promising potential of applying in cell biology and stem-cell science.

  17. Solvothermally Synthesized Sb2Te3 Platelets Show Unexpected Optical Contrasts in Mid-Infrared Near-Field Scanning Microscopy.

    PubMed

    Hauer, Benedikt; Saltzmann, Tobias; Simon, Ulrich; Taubner, Thomas

    2015-05-13

    We report nanoscale-resolved optical investigations on the local material properties of Sb2Te3 hexagonal platelets grown by solvothermal synthesis. Using mid-infrared near-field microscopy, we find a highly symmetric pattern, which is correlated to a growth spiral and which extends over the entire platelet. As the origin of the optical contrast, we identify domains with different densities of charge carriers. On Sb2Te3 samples grown by other means, we did not find a comparable domain structure. PMID:25868047

  18. Combined reflectance confocal microscopy-optical coherence tomography for delineation of basal cell carcinoma margins: an ex vivo study

    NASA Astrophysics Data System (ADS)

    Iftimia, Nicusor; Peterson, Gary; Chang, Ernest W.; Maguluri, Gopi; Fox, William; Rajadhyaksha, Milind

    2016-01-01

    We present a combined reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) approach, integrated within a single optical layout, for diagnosis of basal cell carcinomas (BCCs) and delineation of margins. While RCM imaging detects BCC presence (diagnoses) and its lateral spreading (margins) with measured resolution of ˜1 μm, OCT imaging delineates BCC depth spreading (margins) with resolution of ˜7 μm. When delineating margins in 20 specimens of superficial and nodular BCCs, depth could be reliably determined down to ˜600 μm, and agreement with histology was within about ±50 μm.

  19. Layer-Resolved Evolution of Organic Thin Films Monitored by Photoelectron Emission Microscopy and Optical Reflectance Spectroscopy

    PubMed Central

    2015-01-01

    Photoelectron emission microscopy (PEEM) and differential (optical) reflectance spectroscopy (DRS) have proven independently to be versatile analytical tools for monitoring the evolution of organic thin films during growth. In this paper, we present the first experiment in which both techniques have been applied simultaneously and synchronously. We illustrate how the combined PEEM and DRS results can be correlated to obtain an extended perspective on the electronic and optical properties of a molecular film dependent on the film thickness and morphology. As an example, we studied the deposition of the organic molecule α-sexithiophene on Ag(111) in the thickness range from submonolayers up to several monolayers. PMID:26523159

  20. Tunable Plasmonic Coupling in Self-Assembled Binary Nanocrystal Super lattices Studied by Correlated Optical Microspectrophotometry and Electron Microscopy

    SciTech Connect

    Ye, XC; Chen, J; Diroll, BT; Murray, CB

    2013-03-01

    We study the plasmonic properties of self-assembled binary nanocrystal superlattices (BNSLs) using correlated optical microspectrophotometry and electron microscopy performed on individual BNSL domains. The strength of near-field couplings between adjacent plasmonic nanocrystals (NCs) can be systematically engineered by varying the NC size, composition, and the lattice symmetry of BNSLs, leading to broadband spectral tunability of the collective plasmonic response of BNSLs across the entire visible spectrum. Self-assembled multicomponent NC superlattices represent a versatile platform for the rational design of macroscopic three-dimensional plasmonic metamaterials with emergent optical characteristics.

  1. Optical coherence photoacoustic microscopy (OC-PAM) with an intensity-modulated continuous-wave broadband light source

    NASA Astrophysics Data System (ADS)

    Liu, Xiaojing; Wen, Rong; Li, Yiwen; Jiao, Shuliang

    2016-06-01

    We developed an optical coherence photoacoustic microscopy system using an intensity-modulated continuous-wave superluminescent diode with a center wavelength of 840 nm. The system can accomplish optical coherence tomography (OCT) and photoacoustic microscopy (PAM) simultaneously. Compared to the system with a pulsed light source, this system is able to achieve OCT imaging with quality as high as conventional spectral-domain OCT. Since both of the OCT and PAM images are generated from the same group of photons, they are intrinsically registered in the lateral directions. The system was tested for multimodal imaging the vasculature of mouse ear in vivo by using gold nanorods as contrast agent for PAM, as well as excised porcine eyes ex vivo. The OCT and PAM images showed complimentary information of the sample.

  2. Sub-40 fs, 1060-nm Yb-fiber laser enhances penetration depth in nonlinear optical microscopy of human skin

    NASA Astrophysics Data System (ADS)

    Balu, Mihaela; Saytashev, Ilyas; Hou, Jue; Dantus, Marcos; Tromberg, Bruce J.

    2015-12-01

    Advancing the practical utility of nonlinear optical microscopy requires continued improvement in imaging depth and contrast. We evaluated second-harmonic generation (SHG) and third-harmonic generation images from ex vivo human skin and showed that a sub-40 fs, 1060-nm Yb-fiber laser can enhance SHG penetration depth by up to 80% compared to a >100 fs, 800 nm Ti:sapphire source. These results demonstrate the potential of fiber-based laser systems to address a key performance limitation related to nonlinear optical microscopy (NLOM) technology while providing a low-barrier-to-access alternative to Ti:sapphire sources that could help accelerate the movement of NLOM into clinical practice.

  3. Wide-field optical detection of nanoparticles using on-chip microscopy and self-assembled nanolenses

    NASA Astrophysics Data System (ADS)

    Mudanyali, Onur; McLeod, Euan; Luo, Wei; Greenbaum, Alon; Coskun, Ahmet F.; Hennequin, Yves; Allier, Cédric P.; Ozcan, Aydogan

    2013-03-01

    The direct observation of nanoscale objects is a challenging task for optical microscopy because the scattering from an individual nanoparticle is typically weak at optical wavelengths. Electron microscopy therefore remains one of the gold standard visualization methods for nanoparticles, despite its high cost, limited throughput and restricted field-of-view. Here, we describe a high-throughput, on-chip detection scheme that uses biocompatible wetting films to self-assemble aspheric liquid nanolenses around individual nanoparticles to enhance the contrast between the scattered and background light. We model the effect of the nanolens as a spatial phase mask centred on the particle and show that the holographic diffraction pattern of this effective phase mask allows detection of sub-100 nm particles across a large field-of-view of >20 mm2. As a proof-of-concept demonstration, we report on-chip detection of individual polystyrene nanoparticles, adenoviruses and influenza A (H1N1) viral particles.

  4. Longitudinal correlation properties of an optical field with broad angular and frequency spectra and their manifestation in interference microscopy

    SciTech Connect

    Lyakin, D V; Ryabukho, V P

    2013-10-31

    The results of theoretical and experimental studies of the longitudinal correlation properties of an optical field with broad angular and frequency spectra and manifestations of these properties in interference microscopy are presented. The joint and competitive influence of the angular and frequency spectra of the object-probing field on the longitudinal resolution and on the amplitude of the interference microscope signals from the interfaces between the media inside a multilayer object is demonstrated. The method of compensating the so-called defocusing effect that arises in the interference microscopy using objectives with a large numerical aperture is experimentally demonstrated, which consists in using as a light source in the interference microscope an illuminating interferometer with a frequency-broadband light source. This method of compensation may be used as the basis of simultaneous determination of geometric thickness and refractive index of media forming a multilayer object. (optical fields)

  5. Wide-field optical detection of nanoparticles using on-chip microscopy and self-assembled nanolenses

    PubMed Central

    Mudanyali, Onur; McLeod, Euan; Luo, Wei; Greenbaum, Alon; Coskun, Ahmet F.; Hennequin, Yves; Allier, Cédric P.; Ozcan, Aydogan

    2013-01-01

    The direct observation of nanoscale objects is a challenging task for optical microscopy because the scattering from an individual nanoparticle is typically weak at optical wavelengths. Electron microscopy therefore remains one of the gold standard visualization methods for nanoparticles, despite its high cost, limited throughput and restricted field-of-view. Here, we describe a high-throughput, on-chip detection scheme that uses biocompatible wetting films to self-assemble aspheric liquid nanolenses around individual nanoparticles to enhance the contrast between the scattered and background light. We model the effect of the nanolens as a spatial phase mask centred on the particle and show that the holographic diffraction pattern of this effective phase mask allows detection of sub-100 nm particles across a large field-of-view of >20 mm2. As a proof-of-concept demonstration, we report on-chip detection of individual polystyrene nanoparticles, adenoviruses and influenza A (H1N1) viral particles. PMID:24358054

  6. Evaluating The Gray Scale Response Difference Associated With Bullet Comparisons Using Optical Microscopy

    NASA Astrophysics Data System (ADS)

    Richards, Victoria J.

    captured images that were converted into light contrast profiles, which were a representation of the bullet surface based on light intensities. From these profiles identified peaks and valleys were grouped into resolution bins or segments of a particular length which are used to subdivide the land impression of the bullet and discriminate peaks and valleys. Results from the profile comparison were, The light microscope produced the highest level of correspondence with 100% total peak correspondence for both laterally and rotationally shifted profiles, 330microm and 6° respectively, with a 20microm bin resolution. We concluded from this study that bullet comparison through the use of images captured by optical microscopy is possible. We further suggest that something similar to the Zeiss Universal light microscope with reflected through-the-lens illumination be used for this methodology for bullet analysis and comparison using a bin resolution should be no less than 15 microns. With this microscope the minimum number of images required to account for potential miss-orientation, or bullet profile variations, is 11 images per land impression.

  7. Design, assembly, and optical bench testing of a high-numerical-aperture miniature injection-molded objective for fiber-optic confocal reflectance microscopy

    NASA Astrophysics Data System (ADS)

    Chidley, Matthew D.; Carlson, Kristen D.; Richards-Kortum, Rebecca R.; Descour, Michael R.

    2006-04-01

    The design, analysis, assembly methods, and optical-bench test results for a miniature injection-molded plastic objective lens used in a fiber-optic confocal reflectance microscope are presented. The five-lens plastic objective was tested as a stand-alone optical system before its integration into a confocal microscope for in vivo imaging of cells and tissue. Changing the spacing and rotation of the individual optical elements can compensate for fabrication inaccuracies and improve performance. The system performance of the miniature objective lens is measured by use of an industry-accepted slanted-edge modulation transfer function (MTF) metric. An estimated Strehl ratio of 0.61 and a MTF value of 0.66 at the fiber-optic bundle Nyquist frequency have been obtained. The optical bench testing system is configured to permit interactive optical alignment during testing to optimize performance. These results are part of an effort to demonstrate the manufacturability of low-cost, high-performance biomedical optics for high-resolution in vivo imaging. Disposable endoscopic microscope objectives could help in vivo confocal microscopy technology mature to permit wide-scale clinical screening and detection of early cancers and precancerous lesions.

  8. Specific surface area of snow samples determined by CH4 adsorption at 77 K and estimated by optical microscopy and scanning electron microscopy.

    PubMed

    Dominé, F; Cabanes, A; Taillandier, A S; Legagneux, L

    2001-02-15

    Snow is a divided medium that can adsorb atmospheric trace gases. Evaluating the impact of the snow cover on atmospheric chemistry therefore requires the knowledge of the specific surface area (SSA) of snow. This paper compares the results of three methods used to measure or estimate the SSA of four snow samples: CH4 adsorption at 77 K, optical microscopy (OM), and scanning electron microscopy (SEM, used only on two samples). Within error bars, CH4 adsorption and OM yield similar results on three of the four snow samples. Values for the 4th sample are within a factor of 2. For both samples where CH4 adsorption, OM, and SEM are used, all three methods yield similar results, but CH4 adsorption always has a better accuracy and a much better precision. Thus, despite its ease of use, estimates from OM images are often not accurate enough to monitor the evolution of snow SSA. The main sources of error in the OM method are the difficulty to determine snow crystal thicknesses and to take into account the topography of the snow crystal surface. The combination of CH4 adsorption and OM or SEM can provide useful information on the evolution of both the SSA and the shape of snow crystals. This will be useful to evaluate the respective contributions of adsorption/desorption and sublimation/condensation processes to the impact of the snow cover on atmospheric chemistry. PMID:11349291

  9. Towards phonon photonics: scattering-type near-field optical microscopy reveals phonon-enhanced near-field interaction.

    PubMed

    Hillenbrand, Rainer

    2004-08-01

    Diffraction limits the spatial resolution in classical microscopy or the dimensions of optical circuits to about half the illumination wavelength. Scanning near-field microscopy can overcome this limitation by exploiting the evanescent near fields existing close to any illuminated object. We use a scattering-type near-field optical microscope (s-SNOM) that uses the illuminated metal tip of an atomic force microscope (AFM) to act as scattering near-field probe. The presented images are direct evidence that the s-SNOM enables optical imaging at a spatial resolution on a 10nm scale, independent of the wavelength used (lambda=633 nm and 10 microm). Operating the microscope at specific mid-infrared frequencies we found a tip-induced phonon-polariton resonance on flat polar crystals such as SiC and Si3N4. Being a spectral fingerprint of any polar material such phonon-enhanced near-field interaction has enormous applicability in nondestructive, material-specific infrared microscopy at nanoscale resolution. The potential of s-SNOM to study eigenfields of surface polaritons in nanostructures opens the door to the development of phonon photonics-a proposed infrared nanotechnology that uses localized or propagating surface phonon polaritons for probing, manipulating and guiding infrared light in nanoscale devices, analogous to plasmon photonics. PMID:15231334

  10. Label-free hyperspectral nonlinear optical microscopy of the biofuel micro-algae Haematococcus Pluvialis

    PubMed Central

    Barlow, Aaron M.; Slepkov, Aaron D.; Ridsdale, Andrew; McGinn, Patrick J.; Stolow, Albert

    2014-01-01

    We consider multi-modal four-wave mixing microscopies to be ideal tools for the in vivo study of carotenoid distributions within the important biofuel microalgae Haematococcus pluvialis. We show that hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy generates non-invasive, quantitative real-time concentrations maps of intracellular carotenoid distributions in live algae. PMID:25360358

  11. Label-free hyperspectral nonlinear optical microscopy of the biofuel micro-algae Haematococcus Pluvialis.

    PubMed

    Barlow, Aaron M; Slepkov, Aaron D; Ridsdale, Andrew; McGinn, Patrick J; Stolow, Albert

    2014-10-01

    We consider multi-modal four-wave mixing microscopies to be ideal tools for the in vivo study of carotenoid distributions within the important biofuel microalgae Haematococcus pluvialis. We show that hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy generates non-invasive, quantitative real-time concentrations maps of intracellular carotenoid distributions in live algae. PMID:25360358

  12. Integrated multimodal microscopy, time-resolved fluorescence, and optical-trap rheometry: toward single molecule mechanobiology

    PubMed Central

    Gullapalli, Ramachandra R.; Tabouillot, Tristan; Mathura, Rishi; Dangaria, Jhanvi H.; Butler, Peter J.

    2011-01-01

    Cells respond to forces through coordinated biochemical signaling cascades that originate from changes in single-molecule structure and dynamics and proceed to large-scale changes in cellular morphology and protein expression. To enable experiments that determine the molecular basis of mechanotransduction over these large time and length scales, we construct a confocal molecular dynamics microscope (CMDM). This system integrates total-internal-reflection fluorescence (TIRF), epifluorescence, differential interference contrast (DIC), and 3-D deconvolution imaging modalities with time-correlated single-photon counting (TCSPC) instrumentation and an optical trap. Some of the structures hypothesized to be involved in mechanotransduction are the glycocalyx, plasma membrane, actin cytoskeleton, focal adhesions, and cell-cell junctions. Through analysis of fluorescence fluctuations, single-molecule spectroscopic measurements [e.g., fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence] can be correlated with these subcellular structures in adherent endothelial cells subjected to well-defined forces. We describe the construction of our multimodal microscope in detail and the calibrations necessary to define molecular dynamics in cell and model membranes. Finally, we discuss the potential applications of the system and its implications for the field of mechanotransduction. PMID:17343487

  13. Non-invasive biopsy of doped-ions inside optical substrate by modified two-photon microscopy.

    PubMed

    Wang, Yao-Chin; Chen, Hsiang-Ning; Li, Wang-Yang; Chan, Ming-Che

    2015-01-26

    Doped-ion based optical elements play key roles in optical signal processes, including amplification, absorption, wavelength-filtering, lighting, and polarizing plate. Non-invasively mapping the spatial distribution of the ion concentrations in these optical elements is highly desirable either during the fabrication process or to determine their optical qualities. In this work, we applied modified two-photon fluorescence (m-TPF) microscopy to trace the ion-distributions deep inside the optical elements. For demonstration purposes, polyvinyl alcohol (PVA) polymer films inside polarizing plates are taken as an example, where the spatial distributions of Iodine-dyed ions were measured by the m-TPF microscope in a fast and non-invasive way. The durability of the polarizer films can be distinguished from the axial distribution of the Iodine-dyed ions, without the need to perform a biopsy. This proposed method and demonstrated results show great potential for monitoring the spatial distributions of doped-ions in the optical elements quickly and non-destructively, which would be of great benefit in both scientific research and industrial applications. PMID:25835941

  14. Adaptive optics microscopy enhances image quality in deep layers of CLARITY processed brains of YFP-H mice

    NASA Astrophysics Data System (ADS)

    Reinig, Marc R.; Novack, Samuel W.; Tao, Xiaodong; Ermini, Florian; Bentolila, Laurent A.; Roberts, Dustin G.; MacKenzie-Graham, Allan; Godshalk, S. E.; Raven, M. A.; Kubby, Joel

    2016-03-01

    Optical sectioning of biological tissues has become the method of choice for three-dimensional histological analyses. This is particularly important in the brain were neurons can extend processes over large distances and often whole brain tracing of neuronal processes is desirable. To allow deeper optical penetration, which in fixed tissue is limited by scattering and refractive index mismatching, tissue-clearing procedures such as CLARITY have been developed. CLARITY processed brains have a nearly uniform refractive index and three-dimensional reconstructions at cellular resolution have been published. However, when imaging in deep layers at submicron resolution some limitations caused by residual refractive index mismatching become apparent, as the resulting wavefront aberrations distort the microscopic image. The wavefront can be corrected with adaptive optics. Here, we investigate the wavefront aberrations at different depths in CLARITY processed mouse brains and demonstrate the potential of adaptive optics to enable higher resolution and a better signal-to-noise ratio. Our adaptive optics system achieves high-speed measurement and correction of the wavefront with an open-loop control using a wave front sensor and a deformable mirror. Using adaptive optics enhanced microscopy, we demonstrate improved image quality wavefront, point spread function, and signal to noise in the cortex of YFP-H mice.

  15. Revealing T-Tubules in Striated Muscle with New Optical Super-Resolution Microscopy Techniquess.

    PubMed

    Jayasinghe, Isuru D; Clowsley, Alexander H; Munro, Michelle; Hou, Yufeng; Crossman, David J; Soeller, Christian

    2015-01-01

    The t-tubular system plays a central role in the synchronisation of calcium signalling and excitation-contraction coupling in most striated muscle cells. Light microscopy has been used for imaging t-tubules for well over 100 years and together with electron microscopy (EM), has revealed the three-dimensional complexities of the t-system topology within cardiomyocytes and skeletal muscle fibres from a range of species. The emerging super-resolution single molecule localisation microscopy (SMLM) techniques are offering a near 10-fold improvement over the resolution of conventional fluorescence light microscopy methods, with the ability to spectrally resolve nanometre scale distributions of multiple molecular targets. In conjunction with the next generation of electron microscopy, SMLM has allowed the visualisation and quantification of intricate t-tubule morphologies within large areas of muscle cells at an unprecedented level of detail. In this paper, we review recent advancements in the t-tubule structural biology with the utility of various microscopy techniques. We outline the technical considerations in adapting SMLM to study t-tubules and its potential to further our understanding of the molecular processes that underlie the sub-micron scale structural alterations observed in a range of muscle pathologies. PMID:26913143

  16. Characterization of a Hybrid Optical Microscopy/Laser Ablation Liquid Vortex Capture/Electrospray Ionization System for Mass Spectrometry Imaging

    DOE PAGESBeta

    Cahill, John F.; Kertesz, Vilmos; Van Berkel, Gary J.

    2015-10-22

    Herein, a commercial optical microscope, laser microdissection instrument was coupled with an electrospray ionization mass spectrometer via a low profile liquid vortex capture probe to yield a hybrid optical microscopy/mass spectrometry imaging system. The instrument has bright-field and fluorescence microscopy capabilities in addition to a highly focused UV laser beam that is utilized for laser ablation of samples. With this system, material laser ablated from a sample using the microscope was caught by a liquid vortex capture probe and transported in solution for analysis by electrospray ionization mass spectrometry. Both lane scanning and spot sampling mass spectral imaging modes weremore » used. The smallest area the system was able to ablate was ~0.544 μm × ~0.544 μm, achieved by oversampling of the smallest laser ablation spot size that could be obtained (~1.9 μm). With use of a model photoresist surface, known features as small as ~1.5 μm were resolved. The capabilities of the system with real world samples were demonstrated first with a blended polymer thin film containing poly(2-vinylpyridine) and poly(N-vinylcarbazole). Using spot sampling imaging, sub-micrometer sized features (0.62, 0.86, and 0.98 μm) visible by optical microscopy were clearly distinguished in the mass spectral images. A second real world example showed the imaging of trace amounts of cocaine in mouse brain thin tissue sections. Lastly, with use of a lane scanning mode with ~6 μm × ~6 μm data pixels, features in the tissue as small as 15 μm in size could be distinguished in both the mass spectral and optical images.« less

  17. Characterization of a Hybrid Optical Microscopy/Laser Ablation Liquid Vortex Capture/Electrospray Ionization System for Mass Spectrometry Imaging

    SciTech Connect

    Cahill, John F.; Kertesz, Vilmos; Van Berkel, Gary J.

    2015-10-22

    Herein, a commercial optical microscope, laser microdissection instrument was coupled with an electrospray ionization mass spectrometer via a low profile liquid vortex capture probe to yield a hybrid optical microscopy/mass spectrometry imaging system. The instrument has bright-field and fluorescence microscopy capabilities in addition to a highly focused UV laser beam that is utilized for laser ablation of samples. With this system, material laser ablated from a sample using the microscope was caught by a liquid vortex capture probe and transported in solution for analysis by electrospray ionization mass spectrometry. Both lane scanning and spot sampling mass spectral imaging modes were used. The smallest area the system was able to ablate was ~0.544 μm × ~0.544 μm, achieved by oversampling of the smallest laser ablation spot size that could be obtained (~1.9 μm). With use of a model photoresist surface, known features as small as ~1.5 μm were resolved. The capabilities of the system with real world samples were demonstrated first with a blended polymer thin film containing poly(2-vinylpyridine) and poly(N-vinylcarbazole). Using spot sampling imaging, sub-micrometer sized features (0.62, 0.86, and 0.98 μm) visible by optical microscopy were clearly distinguished in the mass spectral images. A second real world example showed the imaging of trace amounts of cocaine in mouse brain thin tissue sections. Lastly, with use of a lane scanning mode with ~6 μm × ~6 μm data pixels, features in the tissue as small as 15 μm in size could be distinguished in both the mass spectral and optical images.

  18. Functional Expression of Electron Transport Chain and FoF1-ATP Synthase in Optic Nerve Myelin Sheath.

    PubMed

    Bartolucci, Martina; Ravera, Silvia; Garbarino, Greta; Ramoino, Paola; Ferrando, Sara; Calzia, Daniela; Candiani, Simona; Morelli, Alessandro; Panfoli, Isabella

    2015-11-01

    Our previous studies reported evidence for aerobic ATP synthesis by myelin from both bovine brainstem and rat sciatic nerve. Considering that the optic nerve displays a high oxygen demand, here we evaluated the expression and activity of the five Respiratory Complexes in myelin purified from either bovine or murine optic nerves. Western blot analyses on isolated myelin confirmed the expression of ND4L (subunit of Complex I), COX IV (subunit of Complex IV) and β subunit of F1Fo-ATP synthase. Moreover, spectrophotometric and in-gel activity assays on isolated myelin, as well as histochemical activity assays on both bovine and murine transversal optic nerve sections showed that the respiratory Complexes are functional in myelin and are organized in a supercomplex. Expression of oxidative phosphorylation proteins was also evaluated on bovine optic nerve sections by confocal and transmission electron microscopy. Having excluded a mitochondrial contamination of isolated myelin and considering the results form in situ analyses, it is proposed that the oxidative phosphorylation machinery is truly resident in optic myelin sheath. Data may shed a new light on the unknown trophic role of myelin sheath. It may be energy supplier for the axon, explaining why in demyelinating diseases and neuropathies, myelin sheath loss is associated with axonal degeneration. PMID:26334391

  19. Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics.

    PubMed

    Patton, Brian R; Burke, Daniel; Owald, David; Gould, Travis J; Bewersdorf, Joerg; Booth, Martin J

    2016-04-18

    When imaging through tissue, the optical inhomogeneities of the sample generate aberrations that can prevent effective Stimulated Emission Depletion (STED) imaging. This is particularly problematic for 3D-enhanced STED. We present here an adaptive optics implementation that incorporates two adaptive optic elements to enable correction in all beam paths, allowing performance improvement in thick tissue samples. We use this to demonstrate 3D STED imaging of complex structures in Drosophila melanogaster brains. PMID:27137319

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

    PubMed Central

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

    2015-01-01

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

  1. Three-dimensional functional imaging of lung parenchyma using optical coherence tomography combined with confocal fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Gaertner, Maria; Cimalla, Peter; Knels, Lilla; Meissner, Sven; Koch, Edmund

    2011-03-01

    Optical coherence tomography (OCT), as a non-invasive technique for studying tissue morphology, is widely used in in vivo studies, requiring high resolution and fast three-dimensional imaging. Based on light scattering it reveals micrometer sized substructures of the samples due to changes in their optical properties and therefore allows quantification of the specimen's geometry. Utilizing fluorescence microscopy further information can be obtained from molecular compositions embedded in the investigated object. Fluorescent markers, specifically binding to the substance of interest, reveal the sample's chemical structure and give rise to functional studies. This research presents the application of a combined OCT and laser scanning confocal microscopy (LSCM) system to investigate structural details in lung tissue. OCT reveals the three-dimensional morphology of the alveoli whereas fluorescence detection, arising from the fluorophore Sulforhodamin B (SRB) which is binding to elastin, shows the elastic meshwork of the organs extracellular matrix. Different plains of fluorescence can be obtained by using a piezo driven objective and exploiting the confocal functionality of the setup. Both techniques combined in one optical system not only ease the experimental procedure but also contribute to a thorough description of tissue's morphology and chemical composition.

  2. Optics clustered to output unique solutions: a multi-laser facility for combined single molecule and ensemble microscopy.

    PubMed

    Clarke, David T; Botchway, Stanley W; Coles, Benjamin C; Needham, Sarah R; Roberts, Selene K; Rolfe, Daniel J; Tynan, Christopher J; Ward, Andrew D; Webb, Stephen E D; Yadav, Rahul; Zanetti-Domingues, Laura; Martin-Fernandez, Marisa L

    2011-09-01

    Optics clustered to output unique solutions (OCTOPUS) is a microscopy platform that combines single molecule and ensemble imaging methodologies. A novel aspect of OCTOPUS is its laser excitation system, which consists of a central core of interlocked continuous wave and pulsed laser sources, launched into optical fibres and linked via laser combiners. Fibres are plugged into wall-mounted patch panels that reach microscopy end-stations in adjacent rooms. This allows multiple tailor-made combinations of laser colours and time characteristics to be shared by different end-stations minimising the need for laser duplications. This setup brings significant benefits in terms of cost effectiveness, ease of operation, and user safety. The modular nature of OCTOPUS also facilitates the addition of new techniques as required, allowing the use of existing lasers in new microscopes while retaining the ability to run the established parts of the facility. To date, techniques interlinked are multi-photon/multicolour confocal fluorescence lifetime imaging for several modalities of fluorescence resonance energy transfer (FRET) and time-resolved anisotropy, total internal reflection fluorescence, single molecule imaging of single pair FRET, single molecule fluorescence polarisation, particle tracking, and optical tweezers. Here, we use a well-studied system, the epidermal growth factor receptor network, to illustrate how OCTOPUS can aid in the investigation of complex biological phenomena. PMID:21974592

  3. Phase-transition thresholds and vaporization phenomena for ultrasound phase-change nanoemulsions assessed via high-speed optical microscopy.

    PubMed

    Sheeran, Paul S; Matsunaga, Terry O; Dayton, Paul A

    2013-07-01

    Ultrasonically activated phase-change contrast agents (PCCAs) based on perfluorocarbon droplets have been proposed for a variety of therapeutic and diagnostic clinical applications. When generated at the nanoscale, droplets may be small enough to exit the vascular space and then be induced to vaporize with high spatial and temporal specificity by externally-applied ultrasound. The use of acoustical techniques for optimizing ultrasound parameters for given applications can be a significant challenge for nanoscale PCCAs due to the contributions of larger outlier droplets. Similarly, optical techniques can be a challenge due to the sub-micron size of nanodroplet agents and resolution limits of optical microscopy. In this study, an optical method for determining activation thresholds of nanoscale emulsions based on the in vitro distribution of bubbles resulting from vaporization of PCCAs after single, short (<10 cycles) ultrasound pulses is evaluated. Through ultra-high-speed microscopy it is shown that the bubbles produced early in the pulse from vaporized droplets are strongly affected by subsequent cycles of the vaporization pulse, and these effects increase with pulse length. Results show that decafluorobutane nanoemulsions with peak diameters on the order of 200 nm can be optimally vaporized with short pulses using pressures amenable to clinical diagnostic ultrasound machines. PMID:23760161

  4. Optics clustered to output unique solutions: A multi-laser facility for combined single molecule and ensemble microscopy

    NASA Astrophysics Data System (ADS)

    Clarke, David T.; Botchway, Stanley W.; Coles, Benjamin C.; Needham, Sarah R.; Roberts, Selene K.; Rolfe, Daniel J.; Tynan, Christopher J.; Ward, Andrew D.; Webb, Stephen E. D.; Yadav, Rahul; Zanetti-Domingues, Laura; Martin-Fernandez, Marisa L.

    2011-09-01

    Optics clustered to output unique solutions (OCTOPUS) is a microscopy platform that combines single molecule and ensemble imaging methodologies. A novel aspect of OCTOPUS is its laser excitation system, which consists of a central core of interlocked continuous wave and pulsed laser sources, launched into optical fibres and linked via laser combiners. Fibres are plugged into wall-mounted patch panels that reach microscopy end-stations in adjacent rooms. This allows multiple tailor-made combinations of laser colours and time characteristics to be shared by different end-stations minimising the need for laser duplications. This setup brings significant benefits in terms of cost effectiveness, ease of operation, and user safety. The modular nature of OCTOPUS also facilitates the addition of new techniques as required, allowing the use of existing lasers in new microscopes while retaining the ability to run the established parts of the facility. To date, techniques interlinked are multi-photon/multicolour confocal fluorescence lifetime imaging for several modalities of fluorescence resonance energy transfer (FRET) and time-resolved anisotropy, total internal reflection fluorescence, single molecule imaging of single pair FRET, single molecule fluorescence polarisation, particle tracking, and optical tweezers. Here, we use a well-studied system, the epidermal growth factor receptor network, to illustrate how OCTOPUS can aid in the investigation of complex biological phenomena.

  5. Phase-transition thresholds and vaporization phenomena for ultrasound phase-change nanoemulsions assessed via high speed optical microscopy

    PubMed Central

    Sheeran, Paul S.; Matsunaga, Terry O.; Dayton, Paul A.

    2015-01-01

    Ultrasonically activated phase-change contrast agents (PCCAs) based on perfluorocarbon droplets have been proposed for a variety of therapeutic and diagnostic clinical applications. When generated at the nanoscale, droplets may be small enough to exit the vascular space and then be induced to vaporize with high spatial and temporal specificity by externally-applied ultrasound. The use of acoustical techniques for optimizing ultrasound parameters for given applications can be a significant challenge for nanoscale PCCAs due to the contributions of larger outlier droplets. Similarly, optical techniques can be a challenge due to the sub-micron size of nanodroplet agents and resolution limits of optical microscopy. In this study, an optical method for determining activation thresholds of nanoscale emulsions based on the in vitro distribution of bubbles resulting from vaporization of PCCAs after single, short (<10 cycles) ultrasound pulses is evaluated. Through ultra-high-speed microscopy it is shown that the bubbles produced early in the pulse from vaporized droplets are strongly affected by subsequent cycles of the vaporization pulse, and these effects increase with pulse length. Results show that decafluorobutane nanoemulsions with peak diameters on the order of 200 nm can be optimally vaporized with short pulses using pressures amenable to clinical diagnostic ultrasound machines. PMID:23760161

  6. Photoacoustic Microscopy

    PubMed Central

    Yao, Junjie; Wang, Lihong V.

    2012-01-01

    Photoacoustic microscopy (PAM) is a hybrid in vivo imaging technique that acoustically detects optical contrast via the photoacoustic effect. Unlike pure optical microscopic techniques, PAM takes advantage of the weak acoustic scattering in tissue and thus breaks through the optical diffusion limit (~1 mm in soft tissue). With its excellent scalability, PAM can provide high-resolution images at desired maximum imaging depths up to a few millimeters. Compared with backscattering-based confocal microscopy and optical coherence tomography, PAM provides absorption contrast instead of scattering contrast. Furthermore, PAM can image more molecules, endogenous or exogenous, at their absorbing wavelengths than fluorescence-based methods, such as wide-field, confocal, and multi-photon microscopy. Most importantly, PAM can simultaneously image anatomical, functional, molecular, flow dynamic and metabolic contrasts in vivo. Focusing on state-of-the-art developments in PAM, this Review discusses the key features of PAM implementations and their applications in biomedical studies. PMID:24416085

  7. Combined Optical Coherence and Fluorescence Microscopy to assess dynamics and specificity of pancreatic beta-cell tracers

    PubMed Central

    Berclaz, Corinne; Pache, Christophe; Bouwens, Arno; Szlag, Daniel; Lopez, Antonio; Joosten, Lieke; Ekim, Selen; Brom, Maarten; Gotthardt, Martin; Grapin-Botton, Anne; Lasser, Theo

    2015-01-01

    The identification of a beta-cell tracer is a major quest in diabetes research. However, since MRI, PET and SPECT cannot resolve individual islets, optical techniques are required to assess the specificity of these tracers. We propose to combine Optical Coherence Microscopy (OCM) with fluorescence detection in a single optical platform to facilitate these initial screening steps from cell culture up to living rodents. OCM can image islets and vascularization without any labeling. Thereby, it alleviates the need of both genetically modified mice to detect islets and injection of external dye to reveal vascularization. We characterized Cy5.5-exendin-3, an agonist of glucagon-like peptide 1 receptor (GLP1R), for which other imaging modalities have been used and can serve as a reference. Cultured cells transfected with GLP1R and incubated with Cy5.5-exendin-3 show full tracer internalization. We determined that a dose of 1 μg of Cy5.5-exendin-3 is sufficient to optically detect in vivo the tracer in islets with a high specificity. In a next step, time-lapse OCM imaging was used to monitor the rapid and specific tracer accumulation in murine islets and its persistence over hours. This optical platform represents a versatile toolbox for selecting beta-cell specific markers for diabetes research and future clinical diagnosis. PMID:25988507

  8. Wavefront analysis and optimization from conventional liquid crystal displays for low-cost holographic optical tweezers and digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Weber, Andreas; Ortega Clavero, Valentin; Schröder, Werner

    2011-05-01

    In different study fields the manipulation and imaging of micro-sized particles is essential. The use of holographic optical tweezers (HOT) and digital holographic microscopy (DHM) facilitates this task in a non-mechanical way by providing the proper computer generated hologram and the required amount of light. Electrically addressed spatial light modulators (EASLM) found in holographic optical tweezers are typically of the reflective liquid crystal on silicon (LCoS) type which can achieve a phase shift of more than 2π but they are expensive. Similar components like transmissive twisted nematic liquid crystal displays (TN-LCD) are produced in large quantities, their optical characteristics improve rapidly and they are inexpensive. Under certain circumstances these devices can be used instead of expensive spatial light modulators. Consumer grade objectives are not always well corrected for spherical aberration. In that case conventional liquid crystal displays can also compensate these undesired optical effects. For this purpose software-corrected computer generated holograms are calculated. Procedures to analyze and compensate different parameters of a conventional low-cost liquid crystal display, e.g. phase shift evaluation and aberration correction of objectives by Zernike polynomials approximation are explained. The applied software compensation of the computer generated hologram has shown significant improvement of the focus quality. An important price reduction of holographic devices could be achieved by replacing special optical elements if correction algorithms for conventional liquid crystal displays are provided.

  9. Combined Optical Coherence and Fluorescence Microscopy to assess dynamics and specificity of pancreatic beta-cell tracers.

    PubMed

    Berclaz, Corinne; Pache, Christophe; Bouwens, Arno; Szlag, Daniel; Lopez, Antonio; Joosten, Lieke; Ekim, Selen; Brom, Maarten; Gotthardt, Martin; Grapin-Botton, Anne; Lasser, Theo

    2015-01-01

    The identification of a beta-cell tracer is a major quest in diabetes research. However, since MRI, PET and SPECT cannot resolve individual islets, optical techniques are required to assess the specificity of these tracers. We propose to combine Optical Coherence Microscopy (OCM) with fluorescence detection in a single optical platform to facilitate these initial screening steps from cell culture up to living rodents. OCM can image islets and vascularization without any labeling. Thereby, it alleviates the need of both genetically modified mice to detect islets and injection of external dye to reveal vascularization. We characterized Cy5.5-exendin-3, an agonist of glucagon-like peptide 1 receptor (GLP1R), for which other imaging modalities have been used and can serve as a reference. Cultured cells transfected with GLP1R and incubated with Cy5.5-exendin-3 show full tracer internalization. We determined that a dose of 1 μg of Cy5.5-exendin-3 is sufficient to optically detect in vivo the tracer in islets with a high specificity. In a next step, time-lapse OCM imaging was used to monitor the rapid and specific tracer accumulation in murine islets and its persistence over hours. This optical platform represents a versatile toolbox for selecting beta-cell specific markers for diabetes research and future clinical diagnosis. PMID:25988507

  10. The collagen structure of equine articular cartilage characterized using polarization-sensitive optical coherence tomography and non-linear microscopy

    NASA Astrophysics Data System (ADS)

    Mansfield, Jessica C.; Ugryumova, Nadya; Knapp, Karen M.; Matcher, Stephen J.

    2006-09-01

    Equine articular cartilage has been imaged using both polarization-sensitive optical coherence tomography (PS-OCT) and non-linear microscopy. PS-OCT has been used to spatially map the birefringence in the cartilage and we have found that in the vicinity of the lesion the images display a characteristic disruption in the regular birefringence bands shown by normal cartilage. We also note that significant (e.g. x2) variations in the apparent birefringence of samples taken from young (18 month) animals that otherwise appear visually homogeneous are found over spatial scales of a few millimeters. We have also imaged the cartilage using non-linear microscopy and compare the scans taken with second harmonic generation (SHG) light and the two photon fluorescence (TPF) light. SHG images collected using 800 nm excitation reveals the spatial distribution of collagen fibers, whilst TPF images clearly shows the distribution of intracellular and pericellular fluorophores.

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

    NASA Astrophysics Data System (ADS)

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

    2010-09-01

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

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

    PubMed Central

    Bassi, Andrea; Schmid, Benjamin; Huisken, Jan

    2015-01-01

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

  13. Dual-wavelength optical-resolution photoacoustic microscopy for cells with gold nanoparticle bioconjugates in three-dimensional cultures

    NASA Astrophysics Data System (ADS)

    Lee, Po-Yi; Liu, Wei-Wen; Chen, Shu-Ching; Li, Pai-Chi

    2016-03-01

    Three-dimensional (3D) in vitro models bridge the gap between typical two-dimensional cultures and in vivo conditions. However, conventional optical imaging methods such as confocal microscopy and two-photon microscopy cannot accurately depict cellular processing in 3D models due to limited penetration of photons. We developed a dualwavelength optical-resolution photoacoustic microscopy (OR-PAM), which provides sufficient penetration depth and spatial resolution, for studying CD8+ cytotoxic T lymphocytes (CTLs) trafficking in an in vitro 3D tumor microenvironment. CTLs play a cardinal role in host defense against tumor. Efficient trafficking of CTLs to the tumor microenvironment is a critical step for cancer immunotherapy. For the proposed system, gold nanospheres and indocyanine green (ICG) have been remarkable choices for contrast agents for photoacoustic signals due to their excellent biocompatibility and high optical absorption. With distinct absorption spectrums, targeted cells with gold nanospheres and ICG respectively can be identified by switching 523-nm and 800-nm laser irradiation. Moreover, we use an x-y galvanometer scanner to obtain high scanning rate. In the developed system, lateral and axial resolutions were designed at 1.6 μm and 5 μm, respectively. We successfully showed that dual-spectral OR-PAM can map either the distribution of CTLs with gold nanospheres at a visible wavelength of 523 nm or the 3D structure of tumor spheres with ICG in an in vitro 3D microenvironment. Our OR-PAM can provide better biological relevant information in cellular interaction and is potential for preclinical screening of anti-cancer drugs.

  14. Imaging of optical disc using reflection-mode scattering-type scanning near-field optical microscopy.

    PubMed

    Yamaguchi, M; Sasaki, Y; Sasaki, H; Konada, T; Horikawa, Y; Ebina, A; Umezawa, T; Horiguchi, T

    1999-01-01

    A phase-change optical disc was observed using a reflection-mode scattering-type scanning near-field optical microscope (RS-SNOM). In an a.c.-mode SNOM image, the 1.2 microm x 0.6 microm recording marks were successfully observed although the data were recorded on the groove. In contrast, no recording marks could be resolved in a d.c.-mode SNOM image. These results are in good agreement with those from a numerical simulation using the finite difference time domain method. The resolution was better than 100 nm with a.c.-mode SNOM operation and the results indicate that recording marks in phase-change optical media can be directly observed with the RS-SNOM. PMID:11388305

  15. Background-free imaging of plasmonic structures with cross-polarized apertureless scanning near-field optical microscopy.

    PubMed

    Esslinger, M; Dorfmüller, J; Khunsin, W; Vogelgesang, R; Kern, K

    2012-03-01

    We present advances in experimental techniques of apertureless scanning near-field optical microscopy (aSNOM). The rational alignment procedure we outline is based upon a phase singularity that occurs while scanning polarizers around the nominal cross-polarized configuration of s-polarized excitation and p-polarized detection. We discuss the theoretical origin of this topological feature of the setup, which is robust against small deviations, such as minor tip misalignment or shape variations. Setting the polarizers to this singular configuration point eliminates all background signal, allowing for reproducible plasmonic eigenmode mapping with optimal signal-to-noise ratio. PMID:22462926

  16. Analytical model for quantitative prediction of material contrasts in scattering-type near-field optical microscopy.

    PubMed

    Cvitkovic, A; Ocelic, N; Hillenbrand, R

    2007-07-01

    Nanometer-scale mapping of complex optical constants by scattering-type near-field microscopy has been suffering from quantitative discrepancies between the theory and experiments. To resolve this problem, a novel analytical model is presented here. The comparison with experimental data demonstrates that the model quantitatively reproduces approach curves on a Au surface and yields an unprecedented agreement with amplitude and phase spectra recorded on a phonon-polariton resonant SiC sample. The simple closed-form solution derived here should enable the determination of the local complex dielectric function on an unknown sample, thereby identifying its nanoscale chemical composition, crystal structure and conductivity. PMID:19547189

  17. Inverse dynamical photon scattering (IDPS): an artificial neural network based algorithm for three-dimensional quantitative imaging in optical microscopy.

    PubMed

    Jiang, Xiaoming; Van den Broek, Wouter; Koch, Christoph T

    2016-04-01

    Inverse dynamical photon scattering (IDPS), an artificial neural network based algorithm for three-dimensional quantitative imaging in optical microscopy, is introduced. Because the inverse problem entails numerical minimization of an explicit error metric, it becomes possible to freely choose a more robust metric, to introduce regularization of the solution, and to retrieve unknown experimental settings or microscope values, while the starting guess is simply set to zero. The regularization is accomplished through an alternate directions augmented Lagrangian approach, implemented on a graphics processing unit. These improvements are demonstrated on open source experimental data, retrieving three-dimensional amplitude and phase for a thick specimen. PMID:27136994

  18. Fabricating optical lenses by inkjet printing and heat-assisted in situ curing of polydimethylsiloxane for smartphone microscopy

    NASA Astrophysics Data System (ADS)

    Sung, Yu-Lung; Jeang, Jenn; Lee, Chia-Hsiung; Shih, Wei-Chuan

    2015-04-01

    We present a highly repeatable, lithography-free and mold-free method for fabricating flexible optical lenses by in situ curing liquid polydimethylsiloxane droplets on a preheated smooth surface with an inkjet printing process. This method enables us to fabricate lenses with a focal length as short as 5.6 mm, which can be controlled by varying the droplet volume and the temperature of the preheated surface. Furthermore, the lens can be attached to a smartphone camera without any accessories and can produce high-resolution (1 μm) images for microscopy applications.

  19. Multi-Scaled Microstructures in Natural Rubber Characterized by Synchrotron X-ray Scattering and Optical Microscopy

    SciTech Connect

    Toki , S.; Hsiao, B; Amnuaypornsri , S; Sakdapipanich, J; Tanaka, Y

    2008-01-01

    Multi-scaled microstructures induced by natural impurities (i.e., proteins, phospholipids, carbohydrates) in natural rubber (NR) were investigated by synchrotron small-angle X-ray scattering (SAXS), wide-angle X-ray diffraction (WAXD), and optical microscopy using several kinds of untreated and chemically treated un-vulcanized samples. These microstructures include large aggregates (size less than 50 m), well-defined crystals (size less than a few 10 m), and micelles (size much less than 10 m). In un-vulcanized NR samples, even though the concentrations of natural impurities are relatively low, the dispersion of these microstructures significantly affects the mechanical properties

  20. Variable Temperature SiO2 Stripes Spectroscopy Taken by Customized Scattering Type Scanning Near-field Optical Microscopy

    NASA Astrophysics Data System (ADS)

    Li, Chaoran

    Variable temperature scattering-type scanning near-field optical microscopy is the system we built up from basis to explore the phase transition under low temperature. It has advantages of be able to take topography and spectroscopy simultaneously. What the SNOM system measures is the reflective effivient, it is determinde by the dielectric value of the sample, which is an intrinsic chemical propertiy. In this experiment, we taken spectroscopy of a sample with silicon dioxide stripes doped on the silicon substrate, get the contrast of silicon/silison dioxide, which is accord to the prediction of two models. Furthermore, the different contrast under various temperature reveals the temperature dependent dielectric function.

  1. BigNeuron: Large-scale 3D Neuron Reconstruction from Optical Microscopy Images

    PubMed Central

    Peng, Hanchuan; Hawrylycz, Michael; Roskams, Jane; Hill, Sean; Spruston, Nelson; Meijering, Erik; Ascoli, Giorgio A.

    2016-01-01

    Understanding the structure of single neurons is critical for understanding how they function within neural circuits. BigNeuron is a new community effort that combines modern bioimaging informatics, recent leaps in labeling and microscopy, and the widely recognized need for openness and standardization to provide a community resource for automated reconstruction of dendritic and axonal morphology of single neurons. PMID:26182412

  2. Application of Color-TV Microscopy to Self-Taught Optical Mineralogy.

    ERIC Educational Resources Information Center

    Rose, W. I., Jr.; And Others

    1984-01-01

    Describes a self-taught optical mineralogy course which uses eleven 40-60 minute color videocassettes. Laboratory exercises, instructional strategies, and development of graphical manipulation and display exercises are considered. Content of each tape is listed. (BC)

  3. Apollo 11 Lunar Regolith (10084-47) Revisted — A Novel Optical Microscopy Study

    NASA Astrophysics Data System (ADS)

    Greenberg, G.; Kiely, C.; Kiely, C. J.

    2010-03-01

    Using oblique lighting conditions, along with the processing of a through-focal series of frames, has allowed us to obtain high resolution color micrographs of lunar regolith particles showing detail never thought possible with an optical microscope.

  4. A complete digital optics applied to digital holographic microscopy: application to chromatic aberration compensation

    NASA Astrophysics Data System (ADS)

    Colomb, Tristan; Charrière, Florian; Kühn, Jonas; Montfort, Frédéric; Depeursinge, Christian

    2007-06-01

    In optics, optical elements are used to transform, to filter or to process physical wavefronts in order to magnify images, compensate for aberration or to suppress unwanted diffracted order for example. Because digital holography provides numerical wavefronts, we developed a digital optics, involving numerical elements such as numerical lenses and pinholes, to mimic numerically what is usually done physically, with the advantage to be able to define any shape for these elements and to place them everywhere without obstruction problems. We demonstrate that automatic and non-automatic procedures allow diffracted order or parasitic interferences filtering, compensation for aberration and image distortion, and control of position and magnification of reconstructed wavefront. We apply this digital optics to compensate for chromatic aberration in multi-wavelength holography in order to have perfect superposition between wavefronts reconstructed from digital hologram recorded with different wavelengths. This has a great importance for synthetic wavelength digital holography or tomographic digital holography that use multiple wavelengths.

  5. Fiber optic fluorescence microscopy for functional brain imaging in awake, mobile mice

    NASA Astrophysics Data System (ADS)

    Cha, Jaepyeong; Paukert, Martin; Bergles, Dwight E.; Kang, Jin U.

    2014-03-01

    Fiber-optic based optical imaging is an emerging technique for studying brain activity in live animals. Here, we introduce a novel fluorescence fiber-optic microendoscopy approach to minimal invasively detect neural activities in a live mouse brain . The system uses a flexible endoscopic probe composed of a multi-core coherent fiber-bundle terminated with an approximately 1500-micron working distance objective lens. The fiber-optic neural interface is mounted on a 4-mm2 cranial window enabling visualization of glial calcium transients from the same brain region for weeks. We evaluated the system performance through in vivo imaging of GCaMP3 fluorescence in transgenic headrestrained mice during locomotion.

  6. Confocal Optical Imaging Systems and Their Applications in Microscopy and Range Sensing

    NASA Astrophysics Data System (ADS)

    Xiao, Guoqing

    1990-11-01

    Confocal optical imaging systems have been the subject of much recent studies. They have found their applications in biomedical imaging and integrated circuit metrology. Confocal systems differ from the standard optical imaging systems in their use of point illumination and point detection, gaining an improved transverse resolution and superior depth resolution. The depth discrimination capability allows confocal imaging systems to optically cross section translucent objects or to image three-dimensional structures. The improvement in transverse resolution permits them to image structures with more detail and better contrast. This thesis has focused on the design and implementation of the confocal optical imaging systems and their applications. A nonparaxial confocal optical imaging theory is developed based on the scalar Rayleigh-Sommerfeld diffraction theory and Sine Condition without the normally-used thin-lens approximation. Two confocal optical range sensors and a Real-time Confocal Scanning Optical Microscope (RSOM) are demonstrated. It is shown that our RSOM has tremendous advantages over other confocal microscopes both in scanning speed and in the ease of use and alignment. The dependence of the imaging characteristics on the pinhole size and the lens is fully discussed. Experimental measurements are compared with the theoretical calculations. Good agreement is obtained. Also demonstrated in this thesis are numerous applications of the RSOM in integrated circuit metrology and biomedical imaging. Deep trenches as narrow as 1 μm and deep as 6 mu m are observed with the RSOM. The RSOM is not only able to measure the trench depth but, is also able to inspect individual defects inside the trench. Linewidth measurement is also investigated. The RSOM is shown to have an excellent optical cross-sectioning capability. Sectioned images of bones, teeth, and the unprepared cornea of a rabbit eye have been observed. Well-defined sectioned images have been obtained

  7. Imaging of various optical fiber Bragg gratings using differential interference contrast microscopy: analysis and comparison.

    PubMed

    Rollinson, Claire M; Wade, Scott A; Baxter, Greg W; Collins, Stephen F

    2016-02-01

    Differential interference contrast images of various optical fibers and optical fiber Bragg gratings (FBGs), written with the phase mask technique, are presented to provide information about the resultant refractive index variations present in each case. Use of different fiber types using two distinct phase masks producing four Type I FBGs and a Type In FBG allowed similarities and differences in these FBG images due to variations in the Talbot diffraction patterns produced to be studied. PMID:26836080

  8. Near-field penetrating optical microscopy: A live cell nanoscale refractive index measurement technique for quantification of internal macromolecular density

    PubMed Central

    Strasser, Samantha Dale; Shekhawat, Gajendra; Rogers, Jeremy D.; Dravid, Vinayak P.; Taflove, Allen; Backman, Vadim

    2012-01-01

    Quantification of intracellular nanoscale macromolecular density distribution is a fundamental aspect to understanding cellular processes. We report a near-field penetrating optical microscopy (NPOM) technique to directly probe the internal nanoscale macromolecular density of biological cells through quantification of intracellular refractive index (RI). NPOM inserts a tapered optical fiber probe to successive depths into an illuminated sample. A 50 nm diameter probe-tip collects signal that exhibits a linear relationship with the sample RI at a spatial resolution of approximately 50 nm for biologically relevant measurements, one order-of-magnitude finer than the Abbe diffraction limit. Live and fixed cell data illustrate the mechanical ability of a 50 nm probe to penetrate biological samples. PMID:22344088

  9. Near-field penetrating optical microscopy: a live cell nanoscale refractive index measurement technique for quantification of internal macromolecular density.

    PubMed

    Strasser, Samantha Dale; Shekhawat, Gajendra; Rogers, Jeremy D; Dravid, Vinayak P; Taflove, Allen; Backman, Vadim

    2012-02-15

    Quantification of intracellular nanoscale macromolecular density distribution is a fundamental aspect to understanding cellular processes. We report a near-field penetrating optical microscopy (NPOM) technique to directly probe the internal nanoscale macromolecular density of biological cells through quantification of intracellular refractive index (RI). NPOM inserts a tapered optical fiber probe to successive depths into an illuminated sample. A 50 nm diameter probe tip collects signal that exhibits a linear relationship with the sample RI at a spatial resolution of approximately 50 nm for biologically relevant measurements, one order of magnitude finer than the Abbe diffraction limit. Live and fixed cell data illustrate the mechanical ability of a 50 nm probe to penetrate biological samples. PMID:22344088

  10. Near-field scanning optical microscopy cross-sectional measurements of crystalline GaAs solar cells

    SciTech Connect

    Herndon, M. K.; Bradford, W. C.; Collins, R. T.; Hawkins, B. E.; Kuech, T. F.; Friedman, D. J.; Kurtz, S. R.

    2000-07-03

    Near-field scanning optical microscopy (NSOM) was used to study cleaved edges of GaAs solar cell devices. Using visible light for excitation, the NSOM acquired spatially resolved traces of the photocurrent response across the various layers in the device. For excitation energies well above the band gap, carrier recombination at the cleaved surface had a strong influence on the photocurrent signal. Decreasing the excitation energy, which increased the optical penetration depth, allowed the effects of surface recombination to be separated from collection by the pn junction. Using this approach, the NSOM measurements directly observed the effects of a buried minority carrier reflector/passivation layer. (c) 2000 American Institute of Physics.

  11. Concurrent polarization retrieval in multi-heterodyne scanning near-field optical microscopy: validation on silicon form-birefringent grating.

    PubMed

    Yu, L; Sfez, T; Paeder, V; Stenberg, P; Nakagawa, W; Kuittinen, M; Herzig, H P

    2012-10-01

    We demonstrate a concurrent polarization-retrieval algorithm based on a multi-heterodyne scanning near-field optical microscopy (MH-SNOM) measurement system. This method relies on calibration of the polarization properties of the MH-SNOM using an isotropic region of the sample in the vicinity of the nanostructures of interest. We experimentally show the effectiveness of the method on a silicon form-birefringent grating (FBG) with significant polarization diversity. Three spatial dimensional near-field measurements are in agreement with theoretical predictions obtained with rigorous coupled-wave analysis (RCWA). Pseudo-far-field measurements are performed to obtain the effective refractive index of the FBG, emphasizing the validity of the proposed method. This reconstruction algorithm makes the MH-SNOM a powerful tool to analyze concurrently the polarization-dependent near-field optical response of nanostructures with sub wavelength resolution as long as a calibration area is available in close proximity. PMID:23188273

  12. Determination of pigments in colour layers on walls of some selected historical buildings using optical and scanning electron microscopy

    SciTech Connect

    Skapin, A. Sever Ropret, P. Bukovec, P.

    2007-11-15

    For successful restoration of painted walls and painted coloured finishing coats it is necessary to determine the composition of the original colour layers. Identification of the pigments used in The Cistercian Abbey of Sticna and The Manor of Novo Celje was carried out using optical and scanning electron microscopy. Selected samples of wall paintings were inspected by the combined application of an optical microscope and a low-vacuum Scanning Electron Microscope to determine their colour and structural features and to identify the position of individual pigment grains. Energy dispersive spectroscopy was used to determine the elemental distribution on selected surfaces and elemental composition of individual pigments. It was found that the most abundantly used pigments were iron oxide red, cinnabar, green earth, umber, calcium carbonate white, ultramarine, yellow ochre and carbon black. These identifications have allowed us to compare the use of various pigments in buildings from different historical periods.

  13. Real-time monitoring of tumor response to preoperative radiochemotherapy for rectal carcinoma by nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Li, Lianhuang; Chen, Zhifen; Wang, Xingfu; Jiang, Weizhong; Guan, Guoxian; Chen, Jianxin

    2015-03-01

    The continuing advancement of nonlinear optical imaging techniques has opened many new windows in biological exploration. In this work, the nonlinear optical microscopy, based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), was extended to probe tumor response to preoperative radiochemotherapy (RCT) for rectal carcinoma. It was found that MPM has the ability of direct visualization of histopathologic changes in rectal carcinoma following preoperative RCT including stromal fibrosis, colloid response and residual tumors. Our results also showed the capability of MPM using the quantitative analyses of images to quantify these changes. This work may provide the groundwork for further exploration into the application of multiphoton-based endoscopy in a clinical setting.

  14. Establishing the suitability of quantitative optical CT microscopy of PRESAGE® radiochromic dosimeters for the verification of synchrotron microbeam therapy

    NASA Astrophysics Data System (ADS)

    Doran, Simon J.; Rahman, A. T. Abdul; Bräuer-Krisch, Elke; Brochard, Thierry; Adamovics, John; Nisbet, Andrew; Bradley, David

    2013-09-01

    Previous research on optical computed tomography (CT) microscopy in the context of the synchrotron microbeam has shown the potential of the technique and demonstrated high quality images, but has left two questions unanswered: (i) are the images suitably quantitative for 3D dosimetry? and (ii) what is the impact on the spatial resolution of the system of the limited depth-of-field of the microscope optics? Cuvette and imaging studies are reported here that address these issues. Two sets of cuvettes containing the radiochromic plastic PRESAGE® were irradiated at the ID17 biomedical beamline of the European Synchrotron Radiation facility over the ranges 0-20 and 0-35 Gy and a third set of cuvettes was irradiated over the range 0-20 Gy using a standard medical linac. In parallel, three cylindrical PRESAGE® samples of diameter 9.7 mm were irradiated with test patterns that allowed the quantitative capabilities of the optical CT microscope to be verified, and independent measurements of the imaging modulation transfer function (MTF) to be made via two different methods. Both spectrophotometric analysis and imaging gave a linear dose response, with gradients ranging from 0.036-0.041 cm-1 Gy-1 in the three sets of cuvettes and 0.037 (optical CT units) Gy-1 for the imaging. High-quality, quantitative imaging results were obtained throughout the 3D volume, as illustrated by depth-dose profiles. These profiles are shown to be monoexponential, and the linear attention coefficient of PRESAGE® for the synchrotron-generated x-ray beam is measured to be (0.185 ± 0.02) cm-1 in excellent agreement with expectations. Low-level (<5%) residual image artefacts are discussed in detail. It was possible to resolve easily slit patterns of width 37 µm (which are smaller than many of the microbeams used on ID-17), but some uncertainty remains as to whether the low values of MTF for the higher spatial frequencies are scanner related or a result of genuine (but non-ideal) dose

  15. Fabrication and characterization of a silicon cantilever probe with an integrated quartz-glass (fused-silica) tip for scanning near-field optical microscopy.

    PubMed

    Schürmann, G; Noell, W; Staufer, U; de Rooij, N F; Eckert, R; Freyland, J M; Heinzelmann, H

    2001-10-01

    A cantilever-based probe is introduced for use in scanning near-field optical microscopy (SNOM) combined with scanning atomic-force microscopy (AFM). The probes consist of silicon cantilevers with integrated 25-mum-high fused-silica tips. The probes are batch fabricated by microfabrication technology. Transmission electron microscopy reveals that the transparent quartz tips are completely covered with an opaque aluminum layer before the SNOM measurement. Static and dynamic AFM imaging was performed. SNOM imaging in transmission mode of single fluorescent molecules shows an optical resolution better than 32 nm. PMID:18364783

  16. Miniature fiber-optic multiphoton microscopy system using frequency-doubled femtosecond Er-doped fiber laser

    PubMed Central

    Huang, Lin; Mills, Arthur K.; Zhao, Yuan; Jones, David J.; Tang, Shuo

    2016-01-01

    We report on a miniature fiber-optic multiphoton microscopy (MPM) system based on a frequency-doubled femtosecond Er-doped fiber laser. The femtosecond pulses from the laser source are delivered to the miniature fiber-optic probe at 1.58 µm wavelength, where a standard single mode fiber is used for delivery without the need of free-space dispersion compensation components. The beam is frequency-doubled inside the probe by a periodically poled MgO:LiNbO3 crystal. Frequency-doubled pulses at 786 nm with a maximum power of 80 mW and a pulsewidth of 150 fs are obtained and applied to excite intrinsic signals from tissues. A MEMS scanner, a miniature objective, and a multimode collection fiber are further used to make the probe compact. The miniature fiber-optic MPM system is highly portable and robust. Ex vivo multiphoton imaging of mammalian skins demonstrates the capability of the system in imaging biological tissues. The results show that the miniature fiber-optic MPM system using frequency-doubled femtosecond fiber laser can potentially bring the MPM imaging for clinical applications. PMID:27231633

  17. Miniature fiber-optic multiphoton microscopy system using frequency-doubled femtosecond Er-doped fiber laser.

    PubMed

    Huang, Lin; Mills, Arthur K; Zhao, Yuan; Jones, David J; Tang, Shuo

    2016-05-01

    We report on a miniature fiber-optic multiphoton microscopy (MPM) system based on a frequency-doubled femtosecond Er-doped fiber laser. The femtosecond pulses from the laser source are delivered to the miniature fiber-optic probe at 1.58 µm wavelength, where a standard single mode fiber is used for delivery without the need of free-space dispersion compensation components. The beam is frequency-doubled inside the probe by a periodically poled MgO:LiNbO3 crystal. Frequency-doubled pulses at 786 nm with a maximum power of 80 mW and a pulsewidth of 150 fs are obtained and applied to excite intrinsic signals from tissues. A MEMS scanner, a miniature objective, and a multimode collection fiber are further used to make the probe compact. The miniature fiber-optic MPM system is highly portable and robust. Ex vivo multiphoton imaging of mammalian skins demonstrates the capability of the system in imaging biological tissues. The results show that the miniature fiber-optic MPM system using frequency-doubled femtosecond fiber laser can potentially bring the MPM imaging for clinical applications. PMID:27231633

  18. Nanoscale Spatial Organization of Prokaryotic Cells Studied by Super-Resolution Optical Microscopy

    NASA Astrophysics Data System (ADS)

    McEvoy, Andrea Lynn

    All cells spatially organize their interiors, and this arrangement is necessary for cell viability. Until recently, it was believed that only eukaryotic cells spatially segregate their components. However, it is becoming increasingly clear that bacteria also assemble their proteins into complex patterns. In eukaryotic cells, spatial organization arises from membrane bound organelles as well as motor transport proteins which can move cargos within the cell. To date, there are no known motor transport proteins in bacteria and most microbes lack membrane bound organelles, so it remains a mystery how bacterial spatial organization emerges. In hind-sight it is not surprising that bacteria also exhibit complex spatial organization considering much of what we have learned about the basic processes that take place in all cells, such as transcription and translation was first discovered in prokaryotic cells. Perhaps the fundamental principles that govern spatial organization in prokaryotic cells may be applicable in eukaryotic cells as well. In addition, bacteria are attractive model organism for spatial organization studies because they are genetically tractable, grow quickly and much biochemical and structural data is known about them. A powerful tool for observing spatial organization in cells is the fluorescence microscope. By specifically tagging a protein of interest with a fluorescent probe, it is possible to examine how proteins organize and dynamically assemble inside cells. A significant disadvantage of this technology is its spatial resolution (approximately 250 nm laterally and 500 nm axially). This limitation on resolution causes closely spaced proteins to look blurred making it difficult to observe the fine structure within the complexes. This resolution limit is especially problematic within small cells such as bacteria. With the recent invention of new optical microscopies, we now can surpass the existing limits of fluorescence imaging. In some cases, we can

  19. Characterization of X-ray polycapillary optics by LiF crystal radiation detectors through confocal fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Bonfigli, Francesca; Hampai, Dariush; Dabagov, Sultan B.; Montereali, Rosa Maria

    2016-08-01

    Solid-state radiation imaging detectors based on photoluminescent colour centres in lithium fluoride (LiF) crystals have been successfully tested for both advanced 2D and 3D characterizations of X-ray polycapillary optics by a table-top laboratory system. Polycapillary optics can control X-ray beams propagation and allows obtaining quasi-parallel beam (half-lens) or focused beams (full-lens). The combination of a fine-focused micro X-ray tube and a polycapillary lens can provide the high intensity radiation fluxes that are necessary for high resolution X-ray imaging. In this paper we present novel results about advanced characterization of these complex optics by 2D as well as 3D confocal laser fluorescence microscopy of X-ray irradiated LiF crystal detectors. Two dimensional high spatial resolution images on a wide field of view of transmitted X-rays through a semi-lens and 3D direct inspection of the coloured volumes produced in LiF crystals by both focused and parallel X-ray beam transmitted by a full and a semi-lens, respectively, as well as their 3D reconstructions were obtained. The results show that the photoluminescent colour centres volume in LiF crystals combined with an optical sectioning reading system provide information about tomography of transmitted X-ray beams by policapillary optics in a single exposure process. For the first time, the use of LiF crystal plates as versatile radiation imaging luminescent detectors have been used to characterize the operation of polycapillary optics as X-ray lens, in focusing and parallel mode.

  20. A dual-modality optical coherence tomography and fluorescence lifetime imaging microscopy system for simultaneous morphological and biochemical tissue characterization

    PubMed Central

    Park, Jesung; Jo, Javier A.; Shrestha, Sebina; Pande, Paritosh; Wan, Qiujie; Applegate, Brian E.

    2010-01-01

    Most pathological conditions elicit changes in the tissue optical response that may be interrogated by one or more optical imaging modalities. Any single modality typically only furnishes an incomplete picture of the tissue optical response, hence an approach that integrates complementary optical imaging modalities is needed for a more comprehensive non-destructive and minimally-invasive tissue characterization. We have developed a dual-modality system, incorporating optical coherence tomography (OCT) and fluorescence lifetime imaging microscopy (FLIM), that is capable of simultaneously characterizing the 3-D tissue morphology and its biochemical composition. The Fourier domain OCT subsystem, at an 830 nm center wavelength, provided high-resolution morphological volumetric tissue images with an axial and lateral resolution of 7.3 and 13.4 µm, respectively. The multispectral FLIM subsystem, based on a direct pulse-recording approach (upon 355 nm laser excitation), provided two-dimensional superficial maps of the tissue autofluorescence intensity and lifetime at three customizable emission bands with 100 µm lateral resolution. Both subsystems share the same excitation/illumination optical path and are simultaneously raster scanned on the sample to generate coregistered OCT volumes and FLIM images. The developed OCT/FLIM system was capable of a maximum A-line rate of 59 KHz for OCT and a pixel rate of up to 30 KHz for FLIM. The dual-modality system was validated with standard fluorophore solutions and subsequently applied to the characterization of two biological tissue types: postmortem human coronary atherosclerotic plaques, and in vivo normal and cancerous hamster cheek pouch epithelial tissue. PMID:21258457