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Sample records for expressions optical microscopy

  1. Axial Plane Optical Microscopy

    PubMed Central

    Li, Tongcang; Ota, Sadao; Kim, Jeongmin; Wong, Zi Jing; Wang, Yuan; Yin, Xiaobo; Zhang, Xiang

    2014-01-01

    We present axial plane optical microscopy (APOM) that can, in contrast to conventional microscopy, directly image a sample's cross-section parallel to the optical axis of an objective lens without scanning. APOM combined with conventional microscopy simultaneously provides two orthogonal images of a 3D sample. More importantly, APOM uses only a single lens near the sample to achieve selective-plane illumination microscopy, as we demonstrated by three-dimensional (3D) imaging of fluorescent pollens and brain slices. This technique allows fast, high-contrast, and convenient 3D imaging of structures that are hundreds of microns beneath the surfaces of large biological tissues. PMID:25434770

  2. Axial Plane Optical Microscopy

    NASA Astrophysics Data System (ADS)

    Li, Tongcang; Ota, Sadao; Kim, Jeongmin; Wong, Zi Jing; Wang, Yuan; Yin, Xiaobo; Zhang, Xiang

    2014-12-01

    We present axial plane optical microscopy (APOM) that can, in contrast to conventional microscopy, directly image a sample's cross-section parallel to the optical axis of an objective lens without scanning. APOM combined with conventional microscopy simultaneously provides two orthogonal images of a 3D sample. More importantly, APOM uses only a single lens near the sample to achieve selective-plane illumination microscopy, as we demonstrated by three-dimensional (3D) imaging of fluorescent pollens and brain slices. This technique allows fast, high-contrast, and convenient 3D imaging of structures that are hundreds of microns beneath the surfaces of large biological tissues.

  3. Optical imaging. Expansion microscopy.

    PubMed

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

    2015-01-30

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

  4. 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

  5. Trapped particle optical microscopy

    NASA Astrophysics Data System (ADS)

    Malmqvist, Lars; Hertz, Hans M.

    1992-11-01

    A scanned probe optical microscope allowing nondestructive studies of a wide range of objects and surface is described. The microscope utilizes a noninvasive optical trap to position a microscopic probe light source in immediate proximity to the studied object. We demonstrate the method experimentally and show theoretically its potential for optical imaging with subdiffraction limited resolution of, e.g., biological objects.

  6. Quantitative optical phase microscopy.

    PubMed

    Barty, A; Nugent, K A; Paganin, D; Roberts, A

    1998-06-01

    We present a new method for the extraction of quantitative phase data from microscopic phase samples by use of partially coherent illumination and an ordinary transmission microscope. The technique produces quantitative images of the phase profile of the sample without phase unwrapping. The technique is able to recover phase even in the presence of amplitude modulation, making it significantly more powerful than existing methods of phase microscopy. We demonstrate the technique by providing quantitatively correct phase images of well-characterized test samples and show that the results obtained for more-complex samples correlate with structures observed with Nomarski differential interference contrast techniques.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. Disposable optics for microscopy diagnostics.

    PubMed

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

    2015-11-20

    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.

  13. 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.

  14. 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

  15. 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.

  16. Quantitative phase-amplitude microscopy I: optical microscopy.

    PubMed

    Barone-Nugent, E D; Barty, A; Nugent, K A

    2002-06-01

    In this paper, the application of a new optical microscopy method (quantitative phase-amplitude microscopy) to biological imaging is explored, and the issue of resolution and image quality is examined. The paper begins by presenting a theoretical analysis of the method using the optical transfer function formalism of Streibl (1985). The effect of coherence on the formation of the phase image is explored, and it is shown that the resolution of the method is not compromised over that of a conventional bright-field image. It is shown that the signal-to-noise ratio of the phase recovery, however, does depend on the degree of coherence in the illumination. Streibl (1985) notes that partially coherent image formation is a non-linear process because of the intermingling of amplitude and phase information. The work presented here shows that the quantitative phase-amplitude microscopy method acts to linearize the image formation process, and that the phase and amplitude information is properly described using a transfer function analysis. The theoretical conclusions are tested experimentally using an optical microscope and the theoretical deductions are confirmed. Samples for microscopy influence both the phase and amplitude of the light wave and it is demonstrated that the new phase recovery method can separate the amplitude and phase information, something not possible using traditional phase microscopy. In the case of a coherent wave, knowledge of the phase and amplitude constitutes complete information that can be used to emulate other forms of microscopy. This capacity is demonstrated by recovering the phase of a sample and using the data to emulate a differential interference contrast image.

  17. Automated optical microscopy of coated particle fuel

    SciTech Connect

    Kercher, Andrew K; Hunn, John D; Price, Jeffery R; Pappano, Peter J

    2008-01-01

    Fundamental technological advances have occurred during the 20 year hiatus in US research on coated particle nuclear fuel. As part of the recent US Department of Energy s Advanced Gas Reactor Fuel Development and Qualification program, Oak Ridge National Laboratory has utilized advancements in computer automation, digital imaging, and image analysis to modernize US optical microscopy techniques for coated particle nuclear fuel. Automated optical microscopy has enabled detailed and objective analysis of individual particles (hundreds of measurements per particle) and of large sample sizes that far exceed the capabilities of conventional manual microscopy methods (analysis of 1500-5000 particles is common). Demonstrative examples of the capabilities of this automated optical microscopy are given for: (a) shadow imaging of kernels, coated fuel particles, and graphite matrix overcoated particles and (b) cross-sectional analysis of coated fuel particles to determine layer thicknesses.

  18. 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.

  19. 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.

  20. Super-resolution optical microscopy: multiple choices.

    PubMed

    Huang, Bo

    2010-02-01

    The recent invention of super-resolution optical microscopy enables the visualization of fine features in biological samples with unprecedented clarity. It creates numerous opportunities in biology because vast amount of previously obscured subcellular processes now can be directly observed. Rapid development in this field in the past two years offers many imaging modalities that address different needs but they also complicates the choice of the 'perfect' method for answering a specific question. Here I will briefly describe the principles of super-resolution optical microscopy techniques and then focus on comparing their characteristics in various aspects of practical applications. PMID:19897404

  1. 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

  2. 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

  3. 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

  4. 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.

  5. High-Throughput Nonlinear Optical Microscopy

    PubMed Central

    So, Peter T.C.; Yew, Elijah Y.S.; Rowlands, Christopher

    2013-01-01

    High-resolution microscopy methods based on different nonlinear optical (NLO) contrast mechanisms are finding numerous applications in biology and medicine. While the basic implementations of these microscopy methods are relatively mature, an important direction of continuing technological innovation lies in improving the throughput of these systems. Throughput improvement is expected to be important for studying fast kinetic processes, for enabling clinical diagnosis and treatment, and for extending the field of image informatics. This review will provide an overview of the fundamental limitations on NLO microscopy throughput. We will further cover several important classes of high-throughput NLO microscope designs with discussions on their strengths and weaknesses and their key biomedical applications. Finally, this review will close with a perspective of potential future technological improvements in this field. PMID:24359736

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. Optical coherence photoacoustic microscopy: accomplishing optical coherence tomography and photoacoustic microscopy with a single light source

    PubMed Central

    Zhang, Xiangyang; Zhang, Hao F.

    2012-01-01

    Abstract. We developed optical coherence photoacoustic microscopy (OC-PAM) to demonstrate that the functions of optical coherence tomography (OCT) and photoacoustic microscopy (PAM) can be achieved simultaneously by using a single illuminating light source. We used a pulsed broadband laser centered at 580 nm and detected the absorbed photons through photoacoustic detection and the back-scattered photons with an interferometer. In OC-PAM, each laser pulse generates both one OCT A-line and one PAM A-line simultaneously; as a result, the two imaging modalities are intrinsically co-registered in the lateral directions. In vivo images of the mouse ear were acquired to demonstrate the capabilities of OC-PAM. PMID:22502553

  13. 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

  14. [Morton's disease: optic and electron microscopy observations].

    PubMed

    De Palma, L; Tulli, A

    1991-01-01

    The authors performed an optic and electron-microscope investigation above the common digital nerve of the foot, whose fragments had been surgically removed from patients suffering from "Morton metatarsalgia" (neuroma). Histological sections were taken from pre-stenotic swelling in patients with clinical symptoms persisting for one year; perineural thickening without evidence of fibroblastic proliferation could be demonstrated, together with an intraneural deposition of an amorphous substance. In other patients suffering from Morton's disease for a longer time, a more pronounced epineural thickening in the pre-stenotic zone could be shown, with partial replacement of nerve fibers by amorphous substance. In the same patients endoneural fibrositis was seen at the level of the stenosis. Electron-microscopy in patients after one year showed an increase in collagenous endoneural fibers and microfibrils. These histopathological findings suggest a compressive mechanism in the pathogenesis of the damage to the common interdigital nerve in Morton's disease, caused by the extrinsic anatomical structures surrounding the nerve. The so-called "neuroma" can be identified with the pre-stenotic swelling.

  15. 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

  16. Optical-force-induced artifacts in scanning probe microscopy.

    PubMed

    Kohlgraf-Owens, Dana C; Sukhov, Sergey; Dogariu, Aristide

    2011-12-15

    In the practice of near-field scanning probe microscopy, it is typically assumed that the distance regulation is independent of the optical signal. However, we demonstrate that these two signals are entangled due to the inherent action of optically induced force. This coupling leads to artifacts in both estimating the magnitude of optical fields and recording topographic maps.

  17. 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

  18. Optical parametrically gated microscopy in scattering media

    PubMed Central

    Zhao, Youbo; Adie, Steven G.; Tu, Haohua; Liu, Yuan; Graf, Benedikt W.; Chaney, Eric J.; Marjanovic, Marina; Boppart, Stephen A.

    2014-01-01

    High-resolution imaging in turbid media has been limited by the intrinsic compromise between the gating efficiency (removal of multiply-scattered light background) and signal strength in the existing optical gating techniques. This leads to shallow depths due to the weak ballistic signal, and/or degraded resolution due to the strong multiply-scattering background – the well-known trade-off between resolution and imaging depth in scattering samples. In this work, we employ a nonlinear optics based optical parametric amplifier (OPA) to address this challenge. We demonstrate that both the imaging depth and the spatial resolution in turbid media can be enhanced simultaneously by the OPA, which provides a high level of signal gain as well as an inherent nonlinear optical gate. This technology shifts the nonlinear interaction to an optical crystal placed in the detection arm (image plane), rather than in the sample, which can be used to exploit the benefits given by the high-order parametric process and the use of an intense laser field. The coherent process makes the OPA potentially useful as a general-purpose optical amplifier applicable to a wide range of optical imaging techniques. PMID:25321724

  19. 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.

  20. 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.

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

    PubMed

    Miranda, Adelaide; Martins, Marco; De Beule, Pieter A A

    2015-09-01

    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.

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

    PubMed

    Miranda, Adelaide; Martins, Marco; De Beule, Pieter A A

    2015-09-01

    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. PMID:26429446

  3. 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.

  4. Deconvolution in 3-D optical microscopy.

    PubMed

    Shaw, P

    1994-09-01

    Fluorescent probes are becoming ever more widely used in the study of subcellular structure, and determination of their three-dimensional distributions has become very important. Confocal microscopy is now a common technique for overcoming the problem of out-of-focus flare in fluorescence imaging, but an alternative method uses digital image processing of conventional fluorescence images--a technique often termed 'deconvolution' or 'restoration'. This review attempts to explain image deconvolution in a non-technical manner. It is also applicable to 3-D confocal images, and can provide a further significant improvement in clarity and interpretability of such images. Some examples of the application of image deconvolution to both conventional and confocal fluorescence images are shown.

  5. Reconstruction in interferometric synthetic aperture microscopy: comparison with optical coherence tomography and digital holographic microscopy.

    PubMed

    Sheppard, Colin J R; Kou, Shan Shan; Depeursinge, Christian

    2012-03-01

    It is shown that the spatial frequencies recorded in interferometric synthetic aperture microscopy do not correspond to exact backscattering [as they do in unistatic synthetic aperture radar (SAR)] and that the reconstruction process based on SAR is therefore based on an approximation. The spatial frequency response is developed based on the three-dimensional coherent transfer function approach and compared with that in optical coherence tomography and digital holographic microscopy.

  6. Aberrations and adaptive optics in super-resolution microscopy.

    PubMed

    Booth, Martin; Andrade, Débora; Burke, Daniel; Patton, Brian; Zurauskas, Mantas

    2015-08-01

    As one of the most powerful tools in the biological investigation of cellular structures and dynamic processes, fluorescence microscopy has undergone extraordinary developments in the past decades. The advent of super-resolution techniques has enabled fluorescence microscopy - or rather nanoscopy - to achieve nanoscale resolution in living specimens and unravelled the interior of cells with unprecedented detail. The methods employed in this expanding field of microscopy, however, are especially prone to the detrimental effects of optical aberrations. In this review, we discuss how super-resolution microscopy techniques based upon single-molecule switching, stimulated emission depletion and structured illumination each suffer from aberrations in different ways that are dependent upon intrinsic technical aspects. We discuss the use of adaptive optics as an effective means to overcome this problem.

  7. 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.

  8. 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.

  9. 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,…

  10. 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

  11. Understanding the optics to aid microscopy image segmentation.

    PubMed

    Yin, Zhaozheng; Li, Kang; Kanade, Takeo; Chen, Mei

    2010-01-01

    Image segmentation is essential for many automated microscopy image analysis systems. Rather than treating microscopy images as general natural images and rushing into the image processing warehouse for solutions, we propose to study a microscope's optical properties to model its image formation process first using phase contrast microscopy as an exemplar. It turns out that the phase contrast imaging system can be relatively well explained by a linear imaging model. Using this model, we formulate a quadratic optimization function with sparseness and smoothness regularizations to restore the "authentic" phase contrast images that directly correspond to specimen's optical path length without phase contrast artifacts such as halo and shade-off. With artifacts removed, high quality segmentation can be achieved by simply thresholding the restored images. The imaging model and restoration method are quantitatively evaluated on two sequences with thousands of cells captured over several days. PMID:20879233

  12. Assessment of nerve ultrastructure by fibre-optic confocal microscopy.

    PubMed

    Cushway, T R; Lanzetta, M; Cox, G; Trickett, R; Owen, E R

    1996-01-01

    Fibre-optic technology combined with confocality produces a microscope capable of optical thin sectioning. In this original study, tibial nerves have been stained in a rat model with a vital dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide, and analysed by fibre-optic confocal microscopy to produce detailed images of nerve ultrastructure. Schwann cells, nodes of Ranvier and longitudinal myelinated sheaths enclosing axons were clearly visible. Single axons appeared as brightly staining longitudinal structures. This allowed easy tracing of multiple signal axons within the nerve tissue. An accurate measurement of internodal lengths was easily accomplished. This technique is comparable to current histological techniques, but does not require biopsy, thin sectioning or tissue fixing. This study offers a standard for further in vivo microscopy, including the possibility of monitoring the progression of nerve regeneration following microsurgical neurorraphy. PMID:9393664

  13. Gabor-based fusion technique for Optical Coherence Microscopy.

    PubMed

    Rolland, Jannick P; Meemon, Panomsak; Murali, Supraja; Thompson, Kevin P; Lee, Kye-sung

    2010-02-15

    We recently reported on an Optical Coherence Microscopy technique, whose innovation intrinsically builds on a recently reported - 2 microm invariant lateral resolution by design throughout a 2 mm cubic full-field of view - liquid-lens-based dynamic focusing optical probe [Murali et al., Optics Letters 34, 145-147, 2009]. We shall report in this paper on the image acquisition enabled by this optical probe when combined with an automatic data fusion method developed and described here to produce an in-focus high resolution image throughout the imaging depth of the sample. An African frog tadpole (Xenopus laevis) was imaged with the novel probe and the Gabor-based fusion technique, demonstrating subcellular resolution in a 0.5 mm (lateral) x 0.5 mm (axial) without the need, for the first time, for x-y translation stages, depth scanning, high-cost adaptive optics, or manual intervention. In vivo images of human skin are also presented.

  14. 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.

  15. 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.

  16. Near-field optical microscopy of bacteria thin sections

    NASA Astrophysics Data System (ADS)

    Konnov, Nikolai P.; Baiburin, Vil B.; Shcherbakov, Anatolyi A.; Malakhaeva, Alina N.; Volkov, Yuri P.

    1997-12-01

    Whole bacteria as well as thin sections were investigated in our laboratory by means of near field scanning optical microscope (NSOM). The main problem in NSOM operation is a control of distance between microscopy tip and sample surface. The bacteria thin section is a more preferable sample for NSOM investigation because of its flat surface. For increasing of thin sections' image contrast were used different light microscopy stainers (Eosin, Hematoxylin, etc.). We obtained images of thin sections of plague (Y. Pestis EV) and cholera (V. Cholerae). Lateral resolution in the investigation is about 300 angstroms.

  17. 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.

  18. 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

  19. 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.

  20. Extending the functions of scanning near-field optical microscopy

    NASA Astrophysics Data System (ADS)

    Horneber, A.; van den Berg, M.; Rogalski, J.; Swider, K.; Braun, K.; Meixner, M.; Meixner, A. J.; Zhang, D.

    2014-08-01

    Advanced optical setups are continuously developed to gain deeper insight into microscopic matter. In this paper we report the expansion of a home-built parabolic mirror assisted scanning, near-field optical microscope (PMSNOM) by introducing four complementary functions. 1) We integrated a scanning tunneling feedback function in addition to an already existent shear-force feedback control mechanism. Hence a scanning tunneling microscope (STM)-SNOM is realized whose performance will be demonstrated by the tip-enhanced Raman peaks of graphene sheets on a copper substrate. 2) We integrated an ultrafast laser system into the microscope which allows us to combine nonlinear optical microscopy with hyperspectral SNOM imaging. This particular expansion was used to study influences of plasmonic resonances on nonlinear optical properties of metallic nanostructures. 3) We implemented a polarization angle resolved detection technique which enables us to analyze the local structural order of α-sexithiophene (α-6T). 4) We combined scanning photocurrent microscopy with the microscope. This allows us to study morphology related optical (Raman and photoluminescence) and electrical properties of optoelectronic systems. Our work demonstrates the great potential of turning a SNOM into an advanced multifunctional microscope.

  1. 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

  2. Imaging without fluorescence: nonlinear optical microscopy for quantitative cellular imaging.

    PubMed

    Streets, Aaron M; Li, Ang; Chen, Tao; Huang, Yanyi

    2014-09-01

    Quantitative single-cell analysis enables the characterization of cellular systems with a level of detail that cannot be achieved with ensemble measurement. In this Feature we explore quantitative cellular imaging applications with nonlinear microscopy techniques. We first offer an introductory tutorial on nonlinear optical processes and then survey a range of techniques that have proven to be useful for quantitative live cell imaging without fluorescent labels.

  3. Simultaneous photoacoustic and optically mediated ultrasound microscopy: phantom study.

    PubMed

    Subochev, Pavel; Katichev, Alexey; Morozov, Andrey; Orlova, Anna; Kamensky, Vladislav; Turchin, Ilya

    2012-11-15

    An experimental setup for combined photoacoustic (PA) and optically mediated ultrasound (US) microscopy is presented. A spherically focused 35 MHz polyvinylidene fluoride (PVDF) ultrasonic detector with a numerical aperture of 0.28, a focal distance of 9 mm, and a bandwidth (-6 dB level) of 24 MHz was used to obtain PA and US data with a 3 mm imaging depth. A fiber-optic system was employed to deliver laser excitation pulses from a tunable laser to the studied medium. A single optical pulse was used to form both PA and US A-scans. The probing US pulses were generated thermoelastically due to absorption of backscattered laser radiation by the metalized surface of a PVDF film. PMID:23164853

  4. 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

  5. Design of a fiber-optic multiphoton microscopy handheld probe

    PubMed Central

    Zhao, Yuan; Sheng, Mingyu; Huang, Lin; Tang, Shuo

    2016-01-01

    We have developed a fiber-optic multiphoton microscopy (MPM) system with handheld probe using femtosecond fiber laser. Here we present the detailed optical design and analysis of the handheld probe. The optical systems using Lightpath 352140 and 352150 as objective lens were analyzed. A custom objective module that includes Lightpath 355392 and two customized corrective lenses was designed. Their performances were compared by wavefront error, field curvature, astigmatism, F-θ error, and tolerance in Zemax simulation. Tolerance analysis predicted the focal spot size to be 1.13, 1.19 and 0.83 µm, respectively. Lightpath 352140 and 352150 were implemented in experiment and the measured lateral resolution was 1.22 and 1.3 µm, respectively, which matched with the prediction. MPM imaging by the handheld probe were conducted on leaf, fish scale and rat tail tendon. The MPM resolution can potentially be improved by the custom objective module.

  6. Design of a fiber-optic multiphoton microscopy handheld probe

    PubMed Central

    Zhao, Yuan; Sheng, Mingyu; Huang, Lin; Tang, Shuo

    2016-01-01

    We have developed a fiber-optic multiphoton microscopy (MPM) system with handheld probe using femtosecond fiber laser. Here we present the detailed optical design and analysis of the handheld probe. The optical systems using Lightpath 352140 and 352150 as objective lens were analyzed. A custom objective module that includes Lightpath 355392 and two customized corrective lenses was designed. Their performances were compared by wavefront error, field curvature, astigmatism, F-θ error, and tolerance in Zemax simulation. Tolerance analysis predicted the focal spot size to be 1.13, 1.19 and 0.83 µm, respectively. Lightpath 352140 and 352150 were implemented in experiment and the measured lateral resolution was 1.22 and 1.3 µm, respectively, which matched with the prediction. MPM imaging by the handheld probe were conducted on leaf, fish scale and rat tail tendon. The MPM resolution can potentially be improved by the custom objective module. PMID:27699109

  7. Super-resolution optical microscopy of lipid plasma membrane dynamics.

    PubMed

    Eggeling, Christian

    2015-01-01

    Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS, and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS, the STED-FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid-protein interactions and the traditional lipid 'raft' theory. PMID:25658345

  8. Objective-free optical-resolution photoacoustic microscopy.

    PubMed

    Kim, Chulhong; Park, Sungjo; Kim, Jongki; Lee, Sungrae; Lee, Changho; Jeon, Mansik; Kim, Jeehyun; Oh, Kyunghwan

    2013-01-01

    Optical-resolution photoacoustic microscopy (OR-PAM) becomes a premier microscopic imaging tool in biomedicine because it provides agent-free optical absorption information in tissues. By tightly focusing light to a spot, a significantly improved lateral resolution can be achieved in OR-PAM. The focal spot size is typically determined by the numerical aperture of the used objective lens. Here, we demonstrate objective-free OR-PAM using a fiber optic Bessel beam generator. In this approach, no objective lens is required and, beneficially, the complexities of conventional OR-PAM systems can be greatly relieved. We have obtained photoacoustic images of a carbon fiber with a diameter of ∼6  μm, whose lateral resolution was measured to be better than 6 to 7 μm.

  9. 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.

  10. Surface plasmon resonance microscopy: Achieving a quantitative optical response

    NASA Astrophysics Data System (ADS)

    Peterson, Alexander W.; Halter, Michael; Plant, Anne L.; Elliott, John T.

    2016-09-01

    Surface plasmon resonance (SPR) imaging allows real-time label-free imaging based on index of refraction and changes in index of refraction at an interface. Optical parameter analysis is achieved by application of the Fresnel model to SPR data typically taken by an instrument in a prism based figuration. We carry out SPR imaging on a microscope by launching light into a sample and collecting reflected light through a high numerical aperture microscope objective. The SPR microscope enables spatial resolution that approaches the diffraction limit and has a dynamic range that allows detection of subnanometer to submicrometer changes in thickness of biological material at a surface. However, unambiguous quantitative interpretation of SPR changes using the microscope system could not be achieved using the Fresnel model because of polarization dependent attenuation and optical aberration that occurs in the high numerical aperture objective. To overcome this problem, we demonstrate a model to correct for polarization diattenuation and optical aberrations in the SPR data and develop a procedure to calibrate reflectivity to index of refraction values. The calibration and correction strategy for quantitative analysis was validated by comparing the known indices of refraction of bulk materials with corrected SPR data interpreted with the Fresnel model. Subsequently, we applied our SPR microscopy method to evaluate the index of refraction for a series of polymer microspheres in aqueous media and validated the quality of the measurement with quantitative phase microscopy.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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.

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

    PubMed

    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.

  1. Widefield multiphoton microscopy with image-based adaptive optics

    NASA Astrophysics Data System (ADS)

    Chang, C.-Y.; Cheng, L.-C.; Su, H.-W.; Yen, W.-C.; Chen, S.-J.

    2012-10-01

    Unlike conventional multiphoton microscopy according to pixel by pixel point scanning, a widefield multiphoton microscope based on spatiotemporal focusing has been developed to provide fast optical sectioning images at a frame rate over 100 Hz. In order to overcome the aberrations of the widefield multiphoton microscope and the wavefront distortion from turbid biospecimens, an image-based adaptive optics system (AOS) was integrated. The feedback control signal of the AOS was acquired according to locally maximize image intensity, which were provided by the widefield multiphoton excited microscope, by using a hill climbing algorithm. Then, the control signal was utilized to drive a deformable mirror in such a way as to eliminate the aberration and distortion. A R6G-doped PMMA thin film is also increased by 3.7-fold. Furthermore, the TPEF image quality of 1 μm fluorescent beads sealed in agarose gel at different depths is improved.

  2. 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.

  3. 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.

  4. Spiral phase plate contrast in optical and electron microscopy

    NASA Astrophysics Data System (ADS)

    Juchtmans, Roeland; Clark, Laura; Lubk, Axel; Verbeeck, Jo

    2016-08-01

    The use of phase plates in the back focal plane of a microscope is a well-established technique in optical microscopy to increase the contrast of weakly interacting samples and is gaining interest in electron microscopy as well. In this paper we study the spiral phase plate (SPP), also called helical, vortex, or two-dimensional Hilbert phase plate, which adds an angularly dependent phase of the form ei ℓ ϕk to the exit wave in Fourier space. In the limit of large collection angles, we analytically calculate that the average of a pair of ℓ =±1 SPP filtered images is directly proportional to the gradient squared of the exit wave, explaining the edge contrast previously seen in optical SPP work. We discuss the difference between a clockwise-anticlockwise pair of SPP filtered images and derive conditions under which the modulus of the wave's gradient can be seen directly from one SPP filtered image. This work provides the theoretical background to interpret images obtained with a SPP, thereby opening new perspectives for new experiments to study, for example, magnetic materials in an electron microscope.

  5. 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.

  6. Motion Compensation for in vivo Sub-Cellular Optical Microscopy

    PubMed Central

    Lucotte, Bertrand; Balaban, Robert S.

    2014-01-01

    In this review we focus on the impact of tissue motion on attempting to conduct sub-cellular resolution optical microscopy, in vivo. Our position is that tissue motion is one of the major barriers to conducting these studies along with light induced damage, optical probe loading as well as absorbance and scattering effects on the excitation point spread function and collection of emitted light. Recent developments in the speed of image acquisition have reached the limit, in most cases, where the signal from a sub-cellular voxel limits the speed and not the scanning rate of the microscope. Different schemes for compensating for tissue displacements due to rigid body and deformation are presented from tissue restriction, gating, adaptive gating and active tissue tracking. We argue that methods that minimally impact the natural physiological motion of the tissue are desirable since the major reason to perform in vivo studies is to evaluate normal physiological functions. Towards this goal, the methods for active tracking using the optical imaging data itself to monitor tissue displacement and actively move the FOV of the microscope to match the tissue deformation in near real time. Critical for this development was the implementation of near real time image processing in conjunction with the control of the microscope imaging parameters. Clearly the continuing development of methods of motion compensation as well as significant technological solutions to the other barriers to tissue sub-cellular optical imaging in vivo, including optical aberrations and overall signal to noise, will make major contributions to the understanding of cell biology within the body. PMID:24673143

  7. Combining confocal microscopy with precise force-scope optical tweezers

    NASA Astrophysics Data System (ADS)

    Richardson, Andrew C.; Reihani, Nader; Oddershede, Lene B.

    2006-08-01

    We demonstrate an example of 'confocal-tweezers' wherein confocal images and precise optical force measurements, using photodiodes, are obtained simultaneously in the x-y plane without moving the objective lens. The optical trap is produced using a 1.064μm cw laser and is combined with Leica's TCS SP5 broadband confocal microscope to trap and image living cells. The unique method by which the confocal images are created facilitates the acquisition of images in areas far from the trapping location. In addition, because the scanning process involves moving galvanic mirrors independently of the objective, the trap is held stable in position and is not subject to any error in position for the x-y scan. We have successfully trapped and confocally imaged 80nm gold colloids, 150nm gold colloids and 1μm polystyrene beads whilst making quantitative measurements of the force applied by the trap on each bead. To the best of our knowledge this is the first time that anyone has combined precise force measuring optical tweezers with confocal microscopy. We also discuss some of the technical challenges involved in advancing the experimental set up to make quantitative force measurements in combination with 3D stacking. Having proven the potential of this system in 2D, we hope to develop it further to investigate the nano-mechanics of cell division through the attachment of gold beads to fluorescently labelled organelles in S. pombe yeast cells.

  8. 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.

  9. 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

  10. 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

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

    PubMed

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

    2012-05-01

    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.

  12. 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.

  13. Optical interference artifacts in contact atomic force microscopy images.

    PubMed

    Méndez-Vilas, A; González-Martin, M L; Nuevo, M J

    2002-08-01

    Atomic force microscopy images are usually affected by different kinds of artifacts due to either the microscope design and operation mode or external environmental factors. Optical interferences between the laser light reflected off the top of the cantilever and the light scattered by the surface in the same direction is one of the most frequent sources of height artifact in contact (and occasionally non-contact) images. They are present when imaging highly reflective surfaces, or even when imaging non-reflective materials deposited onto reflective ones. In this study interference patterns have been obtained with a highly polished stainless steel planchet. The influence of these artifacts in surface roughness measurements is discussed, and a semi-quantitative method based on the fast Fourier transform technique is proposed to remove the artifacts from the images. This method improves the results obtained by applying the usual flattening routines.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Full-color structured illumination optical sectioning microscopy.

    PubMed

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

    2015-09-29

    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.

  20. Full-color structured illumination optical sectioning microscopy.

    PubMed

    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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. Differential polarization nonlinear optical microscopy with adaptive optics controlled multiplexed beams.

    PubMed

    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.

  6. 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.

  7. 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

  8. 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

  9. Evanescent-wave scattering in near-field optical microscopy.

    PubMed

    Wannemacher, R; Quinten, M; Pack, A

    1999-01-01

    Extended Mie theory is used to investigate the scattering and extinction of evanescent waves by small spherical particles and aggregates of such particles. Metallic, dielectric and metal-coated dielectric particles are taken into consideration. In contrast to plane-wave excitation, p- and s-polarized spectra differ in the case of evanescent waves due to the inherent asymmetry of both polarizations. Furthermore, contributions from higher multipoles are strongly enhanced, compared with plane-wave excitation, and the enhancement factors are polarization dependent. The corresponding changes in the scattering and extinction spectra are most pronounced in cases where higher multipoles exhibit resonances in the spectral range considered. This applies, for example, to morphological resonances of dielectric particles with size parameters > 1. The effect of the surface, where the evanescent wave is generated by total internal reflection, on the scattering and extinction spectra is investigated via numerical field calculations employing the multiple multipole method. In an application to apertureless near-field optical microscopy, the variation of the scattered power is calculated when a silicon particle is scanned across a silver particle in the evanescent field.

  10. Extended focus optical coherence microscopy and fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Villiger, Martin; Blatter, Cedric; Bachmann, Adrian; Lasser, Theo; Leitgeb, Rainer A.

    2008-02-01

    Fourier domain Optical coherence microscopy (FDOCM) offers excellent sensitivity and high axial resolution to image the structure of biological tissue. The depth information is extracted in parallel and allows very high volume acquisition rates. The present system uses a diffractionless beam, produced with an axicon lens, to achieve high lateral resolution all while maintaining an extended depth of field (xf). The xfOCM signal reveals the spatial distribution of changes of the refractive index in the sample that scatter the incident light. To identify and validate the functionality of the observed structures can proof difficult. In this work the xfOCM setup was interfaced with a fluorescent lifetime imaging (FLIM) system, working in the Fourier domain and measuring the phase offset between the modulated excitation signal and the returned fluorescence intensity. Both the fluorescence amplitude and lifetime are retrieved. The amplitude contains important information due to the selective labeling of the tissue. The lifetime is very sensitive to the surrounding environment and varies for different fluorophores, adding further contrast. The xfOCM tomograms and FLIM images are acquired in parallel. A complementary view of the sample is obtained that helps to understand and interpret the xfOCM signal. The lifetime measurement provides further contrast to perform functional imaging on biological samples such as the rat hair follicle.

  11. Optical Force Probe Microscopy of the Pericellular Matrix

    NASA Astrophysics Data System (ADS)

    McLane, Louis T.; Chang, Patrick; Granqvist, Anna; Boehm, Heike; Kramer, Anthony; Curtis, Jennifer E.

    2012-02-01

    The pericellular matrix is a microns-thick grafted polymer film on the surface of cells. Its structure and mechanics influence processes as diverse as filtration, cell adhesion, proliferation, migration, cancer metastasis and possibly mechanotransduction. Optical force probe microscopy enables dynamic and equilibrium measurements of this polymer film on living cells. We show that equilibrium force measurements can be related to the osmotic pressure in the pericellular matrix, leading to a prediction of a spatially varying correlation length (mesh size) profile ranging from ˜100 nm at the cell surface to 1000 nm near the edge of the cell coat. Assuming the film is brush-like, comparison to polymer brush theory provides estimates of the equilibrium brush length and the grafting density at the surface. The equilibrium length is consistent with that observed during dynamic force measurements, and the grafting density is close to that of maximal packing for the large, space filling molecules in the system - i.e. tethered polymers populated by semi-flexible side chains with cross sections ˜80 x 400 nm^2.

  12. Simulation of image formation in x-ray coded aperture microscopy with polycapillary optics.

    PubMed

    Korecki, P; Roszczynialski, T P; Sowa, K M

    2015-04-01

    In x-ray coded aperture microscopy with polycapillary optics (XCAMPO), the microstructure of focusing polycapillary optics is used as a coded aperture and enables depth-resolved x-ray imaging at a resolution better than the focal spot dimensions. Improvements in the resolution and development of 3D encoding procedures require a simulation model that can predict the outcome of XCAMPO experiments. In this work we introduce a model of image formation in XCAMPO which enables calculation of XCAMPO datasets for arbitrary positions of the object relative to the focal plane as well as to incorporate optics imperfections. In the model, the exit surface of the optics is treated as a micro-structured x-ray source that illuminates a periodic object. This makes it possible to express the intensity of XCAMPO images as a convolution series and to perform simulations by means of fast Fourier transforms. For non-periodic objects, the model can be applied by enforcing artificial periodicity and setting the spatial period larger then the field-of-view. Simulations are verified by comparison with experimental data.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

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

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

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

  19. 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.

  20. Visualization of mouse neuronal ganglia infected by Herpes Simplex Virus 1 (HSV-1) using multimodal non-linear optical microscopy.

    PubMed

    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.

  1. 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

  2. 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

  3. 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.

  4. Visualizing Point Defects in Transition-Metal Dichalcogenides Using Optical Microscopy.

    PubMed

    Jeong, Hye Yun; Lee, Si Young; Ly, Thuc Hue; Han, Gang Hee; Kim, Hyun; Nam, Honggi; Jiong, Zhao; Shin, Bong Gyu; Yun, Seok Joon; Kim, Jaesu; Kim, Un Jeong; Hwang, Sungwoo; Lee, Young Hee

    2016-01-26

    While transmission electron microscopy and scanning tunneling microscopy reveal atomic structures of point defect and grain boundary in monolayer transition-metal dichalcogenides (TMDs), information on point defect distribution in macroscale is still not available. Herein, we visualize the point defect distribution of monolayer TMDs using dark-field optical microscopy. This was realized by anchoring silver nanoparticles on defect sites of MoS2 under light illumination. The optical images clearly revealed that the point defect distribution varies with light power and exposure time. The number of silver nanoparticles increased initially and reached a plateau in response to light power or exposure time. The size of silver nanoparticles was a few hundred nanometers in the plateau region as observed using optical microscopy. The measured defect density in macroscale was ∼2 × 10(10) cm(-2), slightly lower than the observed value (4 × 10(11) cm(-2)) from scanning tunneling microscopy. PMID:26645092

  5. 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.

  6. Near-field scanning optical microscopy using polymethylmethacrylate optical fiber probes.

    PubMed

    Chibani, H; Dukenbayev, K; Mensi, M; Sekatskii, S K; Dietler, G

    2010-02-01

    We report the first use of polymethylmethacrylate (PMMA) optical fiber-made probes for scanning near-field optical microscopy (SNOM). The sharp tips were prepared by chemical etching of the fibers in ethyl acetate, and the probes were prepared by proper gluing of sharpened fibers onto the tuning fork in the conditions of the double resonance (working frequency of a tuning fork coincides with the resonance frequency of dithering of the free-standing part of the fiber) reported earlier for the case of glass fibers. Quality factors of the probes in the range 2000-6000 were obtained, which enables the realization of an excellent topographical resolution including state-of-art imaging of single DNA molecules. Near-field optical performance of the microscope is illustrated by the Photon Scanning Tunneling Microscope images of fluorescent beads with a diameter of 100nm. The preparation of these plastic fiber probes proved to be easy, needs no hazardous material and/or procedures, and typical lifetime of a probe essentially exceeds that characteristic for the glass fiber probe. PMID:20022180

  7. 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.

  8. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2012-01-01

    We demonstrate Optical Coherence Microscopy (OCM) for in vivo imaging of the rat cerebral cortex. Imaging does not require addition of dyes or contrast agents, and is achieved through intrinsic scattering contrast and image processing alone. Furthermore, we demonstrate in vivo, quantitative measurements of optical properties and angiography in the rat cerebral cortex. Imaging depths greater than those achieved by conventional two-photon microscopy are demonstrated.

  10. 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.

  11. Quantitative optical microscopy: measurement of cellular biophysical features with a standard optical microscope.

    PubMed

    Phillips, Kevin G; Baker-Groberg, Sandra M; McCarty, Owen J T

    2014-01-01

    We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC) to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using "off-the-shelf" microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) μm in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with

  12. 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.

  13. Real-time optoacoustic brain microscopy with hybrid optical and acoustic resolution

    NASA Astrophysics Data System (ADS)

    Estrada, Héctor; Turner, Jake; Kneipp, Moritz; Razansky, Daniel

    2014-04-01

    Conventional optoacoustic microscopy operates in two distinct modes of optical resolution, for visualization of superficial tissue layers, or acoustic resolution, intended for deep imaging in scattering tissues. Here we introduce a new microscope design with hybrid optical and acoustic resolution, which provides a smooth transition from optical resolution in superficial microscopic imaging to ultrasonic resolution when imaging at greater depths within intensely scattering tissue layers. Experimental validation of the new hybrid optoacoustic microscopy method was performed in phantoms and by means of transcranial mouse brain imaging in vivo.

  14. Adaptive optics confocal microscopy using direct wavefront sensing.

    PubMed

    Tao, Xiaodong; Fernandez, Bautista; Azucena, Oscar; Fu, Min; Garcia, Denise; Zuo, Yi; Chen, Diana C; Kubby, Joel

    2011-04-01

    Optical aberrations due to the inhomogeneous refractive index of tissue degrade the resolution and brightness of images in deep-tissue imaging. We introduce a confocal fluorescence microscope with adaptive optics, which can correct aberrations based on direct wavefront measurements using a Shack-Hartmann wavefront sensor with a fluorescent bead used as a point source reference beacon. The results show a 4.3× improvement in the Strehl ratio and a 240% improvement in the signal intensity for fixed mouse tissues at depths of up to 100 μm.

  15. Confocal microscopy through a multimode fiber using optical correlation

    NASA Astrophysics Data System (ADS)

    Loterie, Damien; Goorden, Sebastianus A.; Psaltis, Demetri; Moser, Christophe

    2015-12-01

    We report on a method to obtain confocal imaging through multimode fibers using optical correlation. First, we measure the fiber's transmission matrix in a calibration step. This allows us to create focused spots at one end of the fiber by shaping the wavefront sent into it from the opposite end. These spots are scanned over a sample, and the light coming back from the sample via the fiber is optically correlated with the input pattern. We show that this achieves spatial selectivity in the detection. The technique is demonstrated on microbeads, a dried epithelial cell, and a cover glass.

  16. 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.

  17. Scanning near-field optical microscopy signal processing and resolution.

    PubMed

    Grosges, Thomas; Barchiesi, Dominique

    2007-04-20

    To increase the signal-to-noise ratio and to remove the spatially slow varying signals, a lock-in amplifier is often used in scanning probe microscopy. The signal reconstructed from the lock-in data contains the contributions of the evanescent and homogeneous waves that are mixed in the near-field zone (i.e., at a very short distance). The resolution is determined and a method is given to suppress the useless background information. Experimental images of nanoparticles are processed.

  18. 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

  19. Nanomechanical Characterization with Near-field Optical Microscopy

    NASA Astrophysics Data System (ADS)

    Ahn, Phillip

    A highly sensitive non-destructive material characterization tool is developed with the goal of measuring the high frequency motion of laser generated ultrasound with nanometer scale lateral spatial resolution. The spatial resolution is achieved through the incorporation of near-field scanning optical microscope (NSOM) techniques, which rely on the measurement of the back scattered near-field light intensity from a illuminated probe-tip placed in close proximity to the sample surface. The weak signal level of the NSOM is enhanced by coupling light to surface plasmon polaritons (SPPs) that are localized at the apex of the probe-tip, and a novel heterodyne demodulation technique is additionally developed for efficient suppression of the high background signal content. A series of near-field imaging experiments along with the theoretical confirmations are provided as a proof of concept to the deep sub-wavelength optical imaging capabilities of the NSOM and the plasmonic nanofocusing probe. The plasmonic near-field scanning optical microscope (p-NSOM) is subsequently used for local detection of the laser generated ultrasound and nanomechanical characterization of doubly clamped resonators. An optoacoustic transducer integrating constrained generation is fabricated, and acoustic waves excited by sub-surface absorption are measured using the plasmonic probe. The p-NSOM is also used for dynamic characterization of nanoelectromechanical systems (NEMS): the heterodyne demodulation approach is utilized in the steady measurement of harmonic vibrations of a NEMS resonator, and laser excitation is used to measure the transient response of the resonator due to a pulsed source in both time and space. These experimental results demonstrate that the p-NSOM is able to measure mechanical motion greater than 100 megahertz and provide a clear indication that the bandwidth of the system is not dependent on the mechanical response of the cantilever probe. This technique, which offers

  20. 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.

  1. 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.

  2. Optical far-field super-resolution microscopy using nitrogen vacancy center ensemble in bulk diamond

    NASA Astrophysics Data System (ADS)

    Li, Shen; Chen, Xiang-dong; Zhao, Bo-Wen; Dong, Yang; Zou, Chong-Wen; Guo, Guang-Can; Sun, Fang-Wen

    2016-09-01

    We demonstrate optical far-field super-resolution microscopy using an array of nitrogen vacancy centers in bulk diamond as near-field optical probes. The local optical field, which transmits through the nanostructures on the diamond surface, is measured by detecting the charge state conversion of the nitrogen vacancy center. Locating the nitrogen vacancy center with a spatial resolution of 6.1 nm is realized with charge state depletion nanoscopy. The nanostructures on the surface of a diamond are then imaged with a resolution below the optical diffraction limit. The results offer an approach to build a general-purpose optical super-resolution microscopy technique and a convenient platform for high spatial resolution quantum sensing with nitrogen vacancy centers.

  3. Advanced Motion Compensation Methods for Intravital Optical Microscopy.

    PubMed

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

    2014-03-01

    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.

  4. 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

  5. In situ microscopy using adjustment-free optics.

    PubMed

    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.

  6. 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.

  7. Adaptive optics applied to coherent anti-Stokes Raman scattering microscopy

    NASA Astrophysics Data System (ADS)

    Girkin, John M.; Poland, Simon P.; Wright, Amanda J.; Freudiger, Christian; Evans, Conor L.; Xie, X. Sunney

    2008-02-01

    We report on the use of adaptive optics in coherent anti-Stokes Raman scattering microscopy (CARS) to improve the image brightness and quality at increased optical penetration depths in biological material. The principle of the technique is to shape the incoming wavefront in such a way that it counteracts the aberrations introduced by imperfect optics and the varying refractive index of the sample. In recent years adaptive optics have been implemented in multiphoton and confocal microscopy. CARS microscopy is proving to be a powerful tool for non-invasive and label-free biomedical imaging with vibrational contrast. As the contrast mechanism is based on a 3 rd order non-linear optical process, it is highly susceptible to aberrations, thus CARS signals are commonly lost beyond the depth of ~100 μm in tissue. We demonstrate the combination of adaptive optics and CARS microscopy for deep-tissue imaging using a deformable membrane mirror. A random search optimization algorithm using the CARS intensity as the figure of merit determined the correct mirror-shape in order to correct for the aberrations. We highlight two different methods of implementation, using a look up table technique and by performing the optimizing in situ. We demonstrate a significant increase in brightness and image quality in an agarose/polystyrene-bead sample and white chicken muscle, pushing the penetration depth beyond 200 μm.

  8. 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.

  9. X-ray microscopy using grazing-incidence reflection optics

    SciTech Connect

    Price, R.H.

    1981-08-06

    The Kirkpatrick-Baez microscopes are described along with their role as the workhorse of the x-ray imaging devices. This role is being extended with the development of a 22X magnification Kirkpatrick-Baez x-ray microscope with multilayer x-ray mirrors. These mirrors can operate at large angles, high x-ray energies, and have a narrow, well defined x-ray energy bandpass. This will make them useful for numerous experiments. However, where a large solid angle is needed, the Woelter microscope will still be necessary and the technology needed to build them will be useful for many other types of x-ray optics.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26306396

  11. 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.

  12. Optical coherence tomography-based freeze-drying microscopy

    PubMed Central

    Mujat, Mircea; Greco, Kristyn; Galbally-Kinney, Kristin L.; Hammer, Daniel X.; Ferguson, R. Daniel; Iftimia, Nicusor; Mulhall, Phillip; Sharma, Puneet; Pikal, Michael J.; Kessler, William J.

    2011-01-01

    A new type of freeze-drying microscope based upon time-domain optical coherence tomography is presented here (OCT-FDM). The microscope allows for real-time, in situ 3D imaging of pharmaceutical formulations in vials relevant for manufacturing processes with a lateral resolution of <7 μm and an axial resolution of <5 μm. Correlation of volumetric structural imaging with product temperature measured during the freeze-drying cycle allowed investigation of structural changes in the product and determination of the temperature at which the freeze-dried cake collapses. This critical temperature is the most important parameter in designing freeze-drying processes of pharmaceutical products. PMID:22254168

  13. 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.

  14. 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.

  15. Direct wavefront sensing in adaptive optical microscopy using backscattered light.

    PubMed

    Rahman, Saad A; Booth, Martin J

    2013-08-01

    Adaptive optics has been used to compensate the detrimental effects of aberrations in a range of high-resolution microscopes. We investigate how backscattered laser illumination can be used as the source for direct wavefront sensing using a pinhole-filtered Shack-Hartmann wavefront sensor. It is found that the sensor produces linear response to input aberrations for a given specimen. The gradient of this response is dependent upon experimental configuration and specimen structure. Cross sensitivity between modes is also observed. The double pass nature of the microscope system leads in general to lower sensitivity to odd-symmetry aberration modes. The results show that there is potential for use of this type of wavefront sensing in microscopes.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. Multicolor stimulated Raman scattering microscopy with a rapidly tunable optical parametric oscillator.

    PubMed

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

    2013-01-15

    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 it sacrifices chemical specificity in samples with overlapping bands compared to broadband (multiplex) excitation. We develop a rapidly tunable picosecond optical parametric oscillator with an electro-optical tunable Lyot filter, and demonstrate multicolor 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.

  3. Follow-up review: recent progress in the development of super-resolution optical microscopy.

    PubMed

    Fujita, Katsumasa

    2016-08-01

    The advent of super-resolution microscopy brought a huge impact to various research fields ranging from the fundamental science to medical and industrial applications. The technological development is still ongoing with involving different scientific disciplines and often changing the standard of optical imaging. In this review, I would like to introduce the recent research progress in super-resolution microscopy as a follow-up for the featured issue in Microscopy (Vol. 64, No. 4, 2015) with discussions especially on the current trends and new directions in the technological development. PMID:27385787

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

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

    PubMed

    Kim, Young Jin; Savukov, Igor

    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 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.

  10. 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

  11. Refractive-index profiling of optical fibers with axial symmetry by use of quantitative phase microscopy.

    PubMed

    Roberts, A; Ampem-Lassen, E; Barty, A; Nugent, K A; Baxter, G W; Dragomir, N M; Huntington, S T

    2002-12-01

    The application of quantitative phase microscopy to refractive-index profiling of optical fibers is demonstrated. Phase images of axially symmetric optical fibers immersed in index-matching fluid are obtained, and the inverse Abel transform is used to obtain the radial refractive-index profile. This technique is straightforward, nondestructive, repeatable, and accurate. Excellent agreement, to within approximately 0.0005, between this method and the index profile obtained with a commercial profiler is obtained.

  12. 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

  13. Understanding the Phase Contrast Optics to Restore Artifact-free Microscopy Images for Segmentation

    PubMed Central

    Yin, Zhaozheng; Kanade, Takeo; Chen, Mei

    2012-01-01

    Phase contrast, a noninvasive microscopy imaging technique, is widely used to capture time-lapse images to monitor the behavior of transparent cells without staining or altering them. Due to the optical principle, phase contrast microscopy images contain artifacts such as the halo and shade-off that hinder image segmentation, a critical step in automated microscopy image analysis. Rather than treating phase contrast microscopy images as general natural images and applying generic image processing techniques on them, we propose to study the optical properties of the phase contrast microscope to model its image formation process. The phase contrast imaging system can be approximated by a linear imaging model. Based on this model and input image properties, we formulate a regularized quadratic cost function to restore artifact-free phase contrast images that directly correspond to the specimen's optical path length. With artifacts removed, high quality segmentation can be achieved by simply thresholding the restored images. The imaging model and restoration method are quantitatively evaluated on microscopy image sequences with thousands of cells captured over several days. We also demonstrate that accurate restoration lays the foundation for high performance in cell detection and tracking. PMID:22386070

  14. Understanding the phase contrast optics to restore artifact-free microscopy images for segmentation.

    PubMed

    Yin, Zhaozheng; Kanade, Takeo; Chen, Mei

    2012-07-01

    Phase contrast, a noninvasive microscopy imaging technique, is widely used to capture time-lapse images to monitor the behavior of transparent cells without staining or altering them. Due to the optical principle, phase contrast microscopy images contain artifacts such as the halo and shade-off that hinder image segmentation, a critical step in automated microscopy image analysis. Rather than treating phase contrast microscopy images as general natural images and applying generic image processing techniques on them, we propose to study the optical properties of the phase contrast microscope to model its image formation process. The phase contrast imaging system can be approximated by a linear imaging model. Based on this model and input image properties, we formulate a regularized quadratic cost function to restore artifact-free phase contrast images that directly correspond to the specimen's optical path length. With artifacts removed, high quality segmentation can be achieved by simply thresholding the restored images. The imaging model and restoration method are quantitatively evaluated on microscopy image sequences with thousands of cells captured over several days. We also demonstrate that accurate restoration lays the foundation for high performance in cell detection and tracking. PMID:22386070

  15. Modulated-alignment dual-axis (MAD) confocal microscopy for deep optical sectioning in tissues

    PubMed Central

    Leigh, Steven Y.; Chen, Ye; Liu, Jonathan T.C.

    2014-01-01

    A strategy is presented to enable optical-sectioning microscopy with improved contrast and imaging depth using low-power (0.5 - 1 mW) diode laser illumination. This technology combines the inherent strengths of focal-modulation microscopy and dual-axis confocal (DAC) microscopy for rejecting out-of-focus and multiply scattered background light in tissues. The DAC architecture is unique in that it utilizes an intersecting pair of illumination and collection beams to improve the spatial-filtering and optical-sectioning performance of confocal microscopy while focal modulation selectively ‘labels’ in-focus signals via amplitude modulation. Simulations indicate that modulating the spatial alignment of dual-axis beams at a frequency f generates signals from the focal volume of the microscope that are modulated at 2f with minimal modulation of background signals, thus providing nearly an order-of-magnitude improvement in optical-sectioning contrast compared to DAC microscopy alone. Experiments show that 2f lock-in detection enhances contrast and imaging depth within scattering phantoms and fresh tissues. PMID:24940534

  16. 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

  17. Optical coherence tomography combined with confocal microscopy for investigation of interfaces in class V cavities

    NASA Astrophysics Data System (ADS)

    Rominu, Mihai; Sinescu, Cosmin; Petrescu, Emanuela; Haiduc, Claudiu; Rominu, Roxana; Enescu, Marius; Hughes, Michael; Bradu, Adrian; Dobre, George; Podoleanu, Adrian G.

    2009-07-01

    Standardized class V cavities, prepared in human extracted teeth, were filled with Premise (Kerr) composite. The specimens were thermo cycled. The interfaces were examined using a system employing two simultaneous imaging channels, an en-face Optical Coherence Tomography channel and a confocal microscopy channel.

  18. 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

  19. DMD-based LED-illumination super-resolution and optical sectioning microscopy.

    PubMed

    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 1.6×10(7) 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.

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Cathodoluminescence-activated nanoimaging: noninvasive near-field optical microscopy in an electron microscope.

    PubMed

    Bischak, Connor G; Hetherington, Craig L; Wang, Zhe; Precht, Jake T; Kaz, David M; Schlom, Darrell G; Ginsberg, Naomi S

    2015-05-13

    We demonstrate a new nanoimaging platform in which optical excitations generated by a low-energy electron beam in an ultrathin scintillator are used as a noninvasive, near-field optical scanning probe of an underlying sample. We obtain optical images of Al nanostructures with 46 nm resolution and validate the noninvasiveness of this approach by imaging a conjugated polymer film otherwise incompatible with electron microscopy due to electron-induced damage. The high resolution, speed, and noninvasiveness of this "cathodoluminescence-activated" platform also show promise for super-resolution bioimaging.

  2. 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.

  3. H-PDLC based waveform controllable optical choppers for FDMF microscopy.

    PubMed

    Zheng, Jihong; Sun, Guoqiang; Jiang, Yanmeng; Wang, Tingting; Huang, Aiqing; Zhang, Yunbo; Tang, Pingyu; Zhuang, Songlin; Liu, Yanjun; Yin, Stuart

    2011-01-31

    An electrically waveform controllable optical chopper based on holographic polymer dispersed liquid crystal grating (H-PDLC) is presented in this paper. The theoretical analyses and experimental results show that the proposed optical chopper has following merits: (1) controllable waveform, (2) no mechanical motion induced vibrational noise, and 
(3) multiple-channel integration capability. The application of this unique electrically controllable optical chopper to frequency division multiplexed fluorescent microscopy is also addressed in this paper, which has the potential to increase the channel capacity, the stability and the reliability. This will be beneficial to the parallel detection, especially for dynamic studies of living biological samples. PMID:21369039

  4. Super-resolution spinning-disk confocal microscopy using optical photon reassignment.

    PubMed

    Azuma, Takuya; Kei, Takayuki

    2015-06-01

    Spinning-disk confocal microscopy is a proven technology for investigating 3D structures of biological specimens. Here we report a super-resolution method based on spinning-disk confocal microscopy that optically improves lateral resolution by a factor of 1.37 with a single exposure. Moreover, deconvolution yields twofold improvement over the diffraction limit. With the help of newly modified Nipkow disk which comprises pinholes and micro-lenses on the front and back respectively, emitted photons from specimen can be optically reassigned to the most probable locations they originate from. Consequently, the improvement in resolution is achieved preserving inherent sectioning capabilities of confocal microscopy. This extremely simple implementation will enable reliable observations at super high resolution in biomedical routine research.

  5. 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

  6. Atomic force microscopy and near-field optical imaging of a spin transition.

    PubMed

    Lopes, Manuel; Quintero, Carlos M; Hernández, Edna M; Velázquez, Víctor; Bartual-Murgui, Carlos; Nicolazzi, William; Salmon, Lionel; Molnár, Gábor; Bousseksou, Azzedine

    2013-09-01

    We report on atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) investigations of single crystals of the spin crossover complex {Fe(pyrazine)[Pt(CN)4]} across the first-order thermal spin transition. We demonstrate for the first time that the change in spin state can be probed with sub-micrometer spatial resolution through various topographic features extracted from AFM data. This original approach based on surface topography analysis should be easy to implement to any phase change material exhibiting sizeable electron-lattice coupling. In addition, AFM images revealed specific topographic features in the crystals, which were correlated with the spatiotemporal evolution of the transition observed by far-field and near-field optical microscopies.

  7. Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy

    PubMed Central

    Yan, Yuling; Petchprayoon, Chutima; Mao, Shu; Marriott, Gerard

    2013-01-01

    Optical switch probes undergo rapid and reversible transitions between two distinct states, one of which may fluoresce. This class of probe is used in various super-resolution imaging techniques and in the high-contrast imaging technique of optical lock-in detection (OLID) microscopy. Here, we introduce optimized optical switches for studies in living cells under standard conditions of cell culture. In particular, a highly fluorescent cyanine probe (Cy or Cy3) is directly or indirectly linked to naphthoxazine (NISO), a highly efficient optical switch that undergoes robust, 405/532 nm-driven transitions between a colourless spiro (SP) state and a colourful merocyanine (MC) state. The intensity of Cy fluorescence in these Cy/Cy3-NISO probes is reversibly modulated between a low and high value in SP and MC states, respectively, as a result of Förster resonance energy transfer. Cy/Cy3-NISO probes are targeted to specific proteins in living cells where defined waveforms of Cy3 fluorescence are generated by optical switching of the SP and MC states. Finally, we introduce a new imaging technique (called OLID-immunofluorescence microscopy) that combines optical modulation of Cy3 fluorescence from Cy3/NISO co-labelled antibodies within fixed cells and OLID analysis to significantly improve image contrast in samples having high background or rare antigens. PMID:23267183

  8. An integrated instrumental setup for the combination of atomic force microscopy with optical spectroscopy.

    PubMed

    Owen, R J; Heyes, C D; Knebel, D; Röcker, C; Nienhaus, G U

    2006-07-01

    In recent years, the study of single biomolecules using fluorescence microscopy and atomic force microscopy (AFM) techniques has resulted in a plethora of new information regarding the physics underlying these complex biological systems. It is especially advantageous to be able to measure the optical, topographical, and mechanical properties of single molecules simultaneously. Here an AFM is used that is especially designed for integration with an inverted optical microscope and that has a near-infrared light source (850 nm) to eliminate interference between the optical experiment and the AFM operation. The Tip Assisted Optics (TAO) system consists of an additional 100 x 100-microm(2) X-Y scanner for the sample, which can be independently and simultaneously used with the AFM scanner. This allows the offset to be removed between the confocal optical image obtained with the sample scanner and the simultaneously acquired AFM topography image. The tip can be positioned exactly into the optical focus while the user can still navigate within the AFM image for imaging or manipulation of the sample. Thus the tip-enhancement effect can be maximized and it becomes possible to perform single molecule manipulation experiments within the focus of a confocal optical image. Here this is applied to simultaneous measurement of single quantum dot fluorescence and topography with high spatial resolution.

  9. 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.

  10. 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

  11. Non-scanning optical near-field microscopy for nanophotonic security

    NASA Astrophysics Data System (ADS)

    Tate, Naoya; Naruse, Makoto; Matsumoto, Tsutomu; Hoga, Morihisa; Ohyagi, Yasuyuki; Nishio, Shumpei; Nomura, Wataru; Ohtsu, Motoichi

    2015-12-01

    We propose a novel method for observing and utilizing nanometrically fluctuating signals due to optical near-field interactions between a probe and target in near-field optical microscopy. Based on a hierarchical structure of the interactions, it is possible to obtain signals that represent two-dimensional spatial patterns without requiring any scanning process. Such signals reveal individual features of each target, and these features, when appropriately extracted and defined, can be used in security applications—an approach that we call nanophotonic security. As an experimental demonstration, output signals due to interactions between a SiO2 probe and Al nanorods were observed by using near-field optical microscopy at a single readout point, and these signals were quantitatively evaluated using an algorithm that we developed for extracting and defining features that can be used for security applications.

  12. 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

  13. 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

  14. 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.

  15. Integrated multimodal optical microscopy for structural and functional imaging of engineered and natural skin

    PubMed Central

    Zhao, Youbo; Graf, Benedikt W.; Chaney, Eric J.; Mahmassani, Ziad; Antoniadou, Eleni; DeVolder, Ross; Kong, Hyunjoon; Boppart, Marni D.; Boppart, Stephen A.

    2015-01-01

    An integrated multimodal optical microscope is demonstrated for high-resolution, structural and functional imaging of engineered and natural skin. This microscope incorporates multiple imaging modalities including optical coherence (OCM), multi-photon (MPM), and fluorescence lifetime imaging microscopy (FLIM), enabling simultaneous visualization of multiple contrast sources and mechanisms from cells and tissues. Spatially co-registered OCM/MPM/FLIM images of multi-layered skin tissues are obtained, which are formed based on complementary information provided by different modalities, i.e., scattering information from OCM, molecular information from MPM, and functional cellular metabolism states from FLIM. Cellular structures in both the dermis and epidermis, especially different morphological and physiological states of keratinocytes from different epidermal layers, are revealed by mutually-validating images. In vivo imaging of human skin is also investigated, which demonstrates the potential of multimodal microscopy for in vivo investigation during engineered skin engraftment. This integrated imaging technique and microscope show the potential for investigating cellular dynamics in developing engineered skin and following in vivo grafting, which will help refine the control and culturing conditions necessary to obtain more robust and physiologically-relevant engineered skin substitutes. Multimodal microscopy images of a microporous 3D hydrogel scaffold seeded with 3T3 fibroblasts. Representative spatially co-registered images were generated based on different methodologies including optical coherence (OCM), multiphoton (MPM), and fluorescence lifetime imaging (FLIM) microscopy. PMID:22371330

  16. 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.

  17. Wide-field two-dimensional multifocal optical-resolution photoacoustic computed microscopy

    PubMed Central

    Xia, Jun; Li, Guo; Wang, Lidai; Nasiriavanaki, Mohammadreza; Maslov, Konstantin; Engelbach, John A.; Garbow, Joel R.; Wang, Lihong V.

    2014-01-01

    Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technique that directly images optical absorption in tissue at high spatial resolution. To date, the majority of OR-PAM systems are based on single focused optical excitation and ultrasonic detection, limiting the wide-field imaging speed. While one-dimensional multifocal OR-PAM (1D-MFOR-PAM) has been developed, the potential of microlens and transducer arrays has not been fully realized. Here, we present the development of two-dimensional multifocal optical-resolution photoacoustic computed microscopy (2D-MFOR-PACM), using a 2D microlens array and a full-ring ultrasonic transducer array. The 10 × 10 mm2 microlens array generates 1800 optical foci within the focal plane of the 512-element transducer array, and raster scanning the microlens array yields optical-resolution photoacoustic images. The system has improved the in-plane resolution of a full-ring transducer array from ≥100 µm to 29 µm and achieved an imaging time of 36 seconds over a 10 × 10 mm2 field of view. In comparison, the 1D-MFOR-PAM would take more than 4 minutes to image over the same field of view. The imaging capability of the system was demonstrated on phantoms and animals both ex vivo and in vivo. PMID:24322226

  18. 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.

  19. X-ray optics for scanning fluorescence microscopy and other applications

    SciTech Connect

    Ryon, R.W.; Warburton, W.K.

    1992-05-01

    Scanning x-ray fluorescence microscopy is analogous to scanning electron microscopy. Maps of chemical element distribution are produced by scanning with a very small x-ray beam. Goal is to perform such scanning microscopy with resolution in the range of <1 to 10 {mu}m, using standard laboratory x-ray tubes. We are investigating mirror optics in the Kirkpatrick-Baez (K-B) configuration. K-B optics uses two curved mirrors mounted orthogonally along the optical axis. The first mirror provides vertical focus, the second mirror provides horizontal focus. We have used two types of mirrors: synthetic multilayers and crystals. Multilayer mirrors are used with lower energy radiation such as Cu K{alpha}. At higher energies such as Ag K{alpha}, silicon wafers are used in order to increase the incidence angles and thereby the photon collection efficiency. In order to increase the surface area of multilayers which reflects x-rays at the Bragg angle, we have designed mirrors with the spacing between layers graded along the optic axis in order to compensate for the changing angle of incidence. Likewise, to achieve a large reflecting surface with silicon, the wafers are placed on a specially designed lever arm which is bent into a log spiral by applying force at one end. In this way, the same diffracting angle is maintained over the entire surface of the wafer, providing a large solid angle for photon collection.

  20. Optical-resolution photoacoustic microscopy based on two-dimensional scanning galvanometer

    NASA Astrophysics Data System (ADS)

    Yuan, Yi; Yang, Sihua; Xing, Da

    2012-01-01

    An optical-resolution photoacoustic microscopy system was designed and fabricated by integration of a two-dimensional scanning galvanometer, an objective lens, an unfocused ultrasound transducer, and a sample stage. The lateral resolution of the system was measured to be ˜500 nm. Ex vivo erythrocytes were used to test the imaging capability of the system, and a single erythrocyte was mapped with high contrast. Furthermore, invivo blood vessels of a mouse ear were clearly shown, and the injured blood vessels were also monitored. The experimental results demonstrate that galvanometer-based photoacoustic microscopy holds clinical potential in detecting lesion of erythrocyte and blood vessel.

  1. 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

  2. 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.

  3. [The presence of asbestos on board ships: optical microscopy as a research instrument].

    PubMed

    Costa, U; Bruni, M; Cimini, F

    1997-01-01

    Identification of asbestos in many different kinds of bulk materials was performed by means of optical microscopy and the results are reported in the present paper. Some hundreds of various samples taken aboard ships were analysed: panels, laggings, spray insulations, etc. These analyses were required to be not only reliable but also rapid. The results demonstrated the high reliability as well as the rapidity of the technique. Using both the well-known dispersion staining technique (with central stop objectives or with dark field condensers) and phase-contrast analyses on the same polarizing microscope, we carried out numerous checks on the optical properties of the fibres. Not only were dispersion staining colours detected but also refractive index, elongation and extinction signs, in order to obtain an absolutely certain identification. The Italian laws which deal with the asbestos detection discourage the use of the optical microscope because of its presumed unreliability. This paper tends to demonstrate that Italian laws underestimate the potential of the optical microscope. Optical microscopy is probably the only technique that is reliable, inexpensive and rapid at the same time.

  4. 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.

  5. 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.

  6. Probing photonic and optoelectronic structures by apertureless scanning near-field optical microscopy.

    PubMed

    Bachelot, Renaud; Lerondel, Gilles; Blaize, Sylvain; Aubert, Sebastien; Bruyant, Aurelien; Royer, Pascal

    2004-08-01

    This report presents the Apertureless Scanning Optical Near-Field Microscope as a powerful tool for the characterization of modern optoelectronic and photonic components with sub-wavelength resolution. We present an overview of the results we obtained in our laboratory over the past few years. By significant examples, it is shown that this specific probe microscopy allows for in situ local quantitative study of semiconductor lasers in operation, integrated optical waveguides produced by ion exchange (single channel or Y junction), and photonic structures.

  7. 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.

  8. Dynamic Light Scattering Microscopy. A Novel Optical Technique to Image Submicroscopic Motions. I: Theory

    PubMed Central

    Dzakpasu, Rhonda; Axelrod, Daniel

    2004-01-01

    The theoretical basis of an optical microscope technique to image dynamically scattered light fluctuation decay rates (dynamic light scattering microscopy) is developed. It is shown that relative motions between scattering centers even smaller than the optical resolution of the microscope are sufficient to produce significant phase variations resulting in interference intensity fluctuations in the image plane. The timescale and time dependence for the temporal autocorrelation function of these intensity fluctuations is derived. The spatial correlation distance, which reports the average distance between constructive and destructive interference in the image plane, is calculated and compared with the pixel size, and the distance dependence of the spatial correlation function is derived. The accompanying article in this issue describes an experimental implementation of dynamic light scattering microscopy. PMID:15298930

  9. 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.

  10. Micromachined solid immersion lenses and optical antennas for scanning near-field optical microscopy

    NASA Astrophysics Data System (ADS)

    Crozier, Kenneth Brian

    The optical microscope is a powerful and ubiquitous measurement and observation tool in science, medicine and industry. In spite of this, however, the resolving power of the optical microscope is fundamentally limited by diffraction. In this work we demonstrate two methods to overcome this limitation based on micromachined Solid Immersion Lenses (SILs) and optical antennas. In the first method for improving optical resolution, the Solid Immersion Lens (SIL), light is focused in a high refractive index lens held close to the sample. Silicon nitride SILs with diameters of 7 micron integrated with atomic force microscope cantilevers are fabricated by surface micromachining. A scanning optical microscope based on the micromachined SIB and operating in reflection and transmission modes at a wavelength of 400nm is presented. The full width at half maximum spot size of the SIL-based microscope is measured to be ˜130nm, which is ˜1.9 times better than the spot size without the SIL (256nm). Furthermore, the optical transmission efficiency of the SIL is ˜64% (with losses due to reflection and absorption), which is significantly better than that of the tapered fiber nearfield scanning optical microscope (typically ˜0.001--0.01%). The second method for improving resolution uses antennas operating at optical wavelengths to enhance the optical fields in regions whose dimensions are much smaller than the wavelength. We present a numerical study based on the finite difference time domain (FDTD) technique, showing that the optical intensity is enhanced by three orders of magnitude in a region whose dimensions are less than ˜lambda/40. A study on the factors influencing intensity enhancement is presented. Optical antennas operating at infrared wavelengths (˜2--10 micron) are fabricated by electron-beam lithography. Far-field measurements on the optical antennas are carried out and found to be in good agreement with FDTD calculations. Lastly, we present a technique in which the

  11. 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.

  12. 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

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

    PubMed

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

    2014-03-01

    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.

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

    PubMed

    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

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

    PubMed

    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.

  16. 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

  17. 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

  18. 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.

  19. Imaging fenestrations in liver sinusoidal endothelial cells by optical localization microscopy.

    PubMed

    Mönkemöller, Viola; Schüttpelz, Mark; McCourt, Peter; Sørensen, Karen; Smedsrød, Bård; Huser, Thomas

    2014-06-28

    Liver sinusoidal endothelial cells (LSEC) are an important class of endothelial cells facilitating the translocation of lipoproteins and small molecules between the liver and blood. A number of clinical conditions, especially metabolic and aging-related disorders, are implicated by improper function of LSECs. Despite their importance, research into these cells is limited because the primary ultrastructures involved in their function are transcellular pores, called fenestrations, with diameters in a size range between 50-200 nm, i.e. well below the optical diffraction limit. Here, we show that we are able to resolve fenestrations with a spatial resolution of ∼20 nm by direct stochastic optical reconstruction microscopy (dSTORM). The cellular plasma membrane was labeled at high fluorophore density with CellMask Deep Red and imaged using a reducing buffer system. We compare the higher degree of structural detail that dSTORM provides to results obtained by 3D structured illumination microscopy (3D-SIM). Our results open up a path to image these physiologically important cells in vitro using highly resolving localization microscopy techniques that could be implemented on non-specialized fluorescence microscopes, enabling their investigation in most biomedical laboratories without the need for electron microscopy.

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. In situ observation of elementary growth processes of protein crystals by advanced optical microscopy.

    PubMed

    Sazaki, Gen; Van Driessche, Alexander E S; Dai, Guoliang; Okada, Masashi; Matsui, Takuro; Otálora, Fermin; Tsukamoto, Katsuo; Nakajima, Kazuo

    2012-07-01

    To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined.

  5. 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

  6. 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

  7. 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

  8. 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.

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

    PubMed Central

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

    2015-01-01

    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

  10. Ultrasharp carbon whisker optical fiber probes for scanning near-field optical microscopy

    NASA Astrophysics Data System (ADS)

    Mensi, Mounir; Mikhailov, Gennadii; Pyatkin, Sergey; Adamcik, Jozef; Sekatskii, Sergey; Dietler, Giovanni

    2010-05-01

    We report the growth of ultrasharp carbon whiskers onto apertured near-field optical glass fiber probes. The ultrasharp carbon whiskers are produced by the electron-assisted dissociation of residual oil vapors present in the vacuum chamber during the electron beam exposition of the tip. This cost effective manufacturing procedure is reproducible, fast and allows controlling the shape of the carbon whisker. The radius of curvature of the whisker apex is approximately 10 nm while its small total length is around 100 nm thus fulfilling the requirements of aperture Scanning Near-Field Optical Microscope (SNOM) probes, i.e. to keep the distance between the sample and the optical aperture during the scanning at subwavelength scale. Furthermore, due to the intrinsic properties of the amorphous carbon whisker, the probes are durable. The carbon whisker optical fiber probes are mounted on tuning-forks using the earlier discussed double-resonant principle. This process ensures a high quality factor of the sensor in the range 2000-5500, which enables to cope with the large stiffness of the tuning-fork actuator and obtain a characteristic noise-limited sensitivity smaller than 10pN necessary to image soft biological samples without destroying them. To illustrate the sensor's performances, transmission near-field optical images of SNOM calibration grating as well as high-resolution state-of-the-art topographic images of single DNA molecules are presented. Prospects of further improvements of the fabrication method enabling to achieve the lighting rod enhancement of the optical near-field (nano-antenna effect) are briefly discussed.

  11. Monitoring the healing process of laser-induced microvascular lesions using optical-resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

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

    2009-02-01

    Optical resolution photoacoustic microscopy (OR-PAM) possesses optical resolution and reveals endogenous optical absorption contrast, promising to be a valuable tool for in vivo microvascular imaging. In laser dermatology, OR-PAM can provide fruitful structural and functional information about the targeted microvascular lesions, such as their threedimensional (3D) morphology, precise location inside the tissue, and blood oxygenation within single vessels, which will facilitate accurate diagnosis and proper treatment. More importantly, the advantages of noninvasiveness and measurement consistency also permit OR-PAM to monitor the healing process of the laser-surgical wound noninvasively. In this work, we employed OR-PAM to monitor the healing process of microvascular lesions induced by nanosecond-pulsed laser. Our results indicate that OR-PAM could be a very useful tool in laser dermatology and laser microsurgery.

  12. High resolution capabilities of all-silica cantilevered probes for near-field optical microscopy.

    PubMed

    Descrovi, Emiliano; Aeschimann, Laure; Soboleva, Irina; De Angelis, Francesco; Giorgis, Fabrizio; Di Fabrizio, Enzo

    2009-11-01

    We report on the possibility of performing Near-field Scanning Optical Microscopy in illumination mode by means of microfabricated, metal-coated silica probes based on transparent cantilevers. A low spring constant silica cantilever hosts a silica tip at its end showing an hyperbolic profile and a circular symmetry. After evaporation of 100 nm of aluminium on the tip and the cantilever we processed the tip apex by means of a FIB, thus obtaining either a probe apex with an optical aperture or an apertureless probe having a thin metal layer on the top. An excellent quality of near-field images of samples showing sub-wavelength features is obtained in both case. In particular, the apertureless probe allows highly resolved topographical and optical images to be collected at the same time. This work further demonstrates that the use of completely transparent, metal-coated cantilevers greatly simplify the light injection into the probe and the fabrication process consequently.

  13. 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

  14. 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

  15. Imaging single chiral nanoparticles in turbid media using circular-polarization optical coherence microscopy.

    PubMed

    Zhang, Pengfei; Mehta, Kalpesh; Rehman, Shakil; Chen, Nanguang

    2014-05-15

    Optical coherence tomography (OCT) is a widely used structural imaging method. However, it has limited use in molecular imaging due to the lack of an effective contrast mechanism. Gold nanoparticles have been widely used as molecular probes for optical microcopy based on Surface Plasmon Resonance (SPR). Unfortunately, the SPR enhanced backscattering from nanoparticles is still relatively weak compared with the background signal from microscopic structures in biological tissues when imaged with OCT. Consequently, it is extremely challenging to perform OCT imaging of conventional nanoparticles in thick tissues with sensitivity comparable to that of fluorescence imaging. We have discovered and demonstrated a novel approach towards remarkable contrast enhancement, which is achieved by the use of a circular-polarization optical coherence microscopy system and 3-dimensional chiral nanostructures as contrast agents. By detecting the circular intensity differential depolarization (CIDD), we successfully acquired high quality images of single chiral nanoparticles underneath a 1-mm-thick tissue -mimicking phantom.

  16. 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

  17. 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.

  18. Rotational Analysis of Spherical, Optically Anisotropic Janus Particles by Dynamic Microscopy.

    PubMed

    Wittmeier, Andrew; Holterhoff, Andrew Leeth; Johnson, Joel; Gibbs, John G

    2015-09-29

    We analyze the rotational dynamics of spherical colloidal Janus particles made from silica (SiO2) with a hemispherical gold/palladium (Au/Pd) cap. Since the refractive index difference between the surrounding fluid and a two-faced, optically anisotropic Janus microsphere is a function of the particle's orientation, it is possible to observe its rotational dynamics with bright-field optical microscopy. We investigate rotational diffusion and constant rotation of single Janus microspheres which are partially tethered to a solid surface so they are free to rotate but show little or no translational motion. Also, since the metal cap is a powerful catalyst in the breakdown of hydrogen peroxide, H2O2, the particles can be activated chemically. In this case, we analyze the motion of coupled Janus dimers which undergo a stable rotary motion about a mutual center. The analysis of both experimental and simulation data, which are microscopy and computer-generated videos, respectively, is based upon individual particle tracking and differential dynamic microscopy (DDM). DDM, which typically requires ensemble averages to extract meaningful information for colloidal dynamics, can be effective in certain situations for systems consisting of single entities. In particular, when translational motion is suppressed, both rotational diffusion and constant rotation can be probed.

  19. Optical and scanning electron microscopy in the single osteoclast resorption assay.

    PubMed

    Boyde, A; Ali, N N; Jones, S J

    1985-01-01

    The present studies relate to the single or isolated osteoclastic resorption function assay which we introduced in 1983 to overcome objections to assays based upon measurements of calcium release from bones, in which it was never strictly controlled whether the mechanism involved the destruction of bone with the formation of classical Howship's lacunae. The method may prove to be quite popular in the near future and has already been adopted by other research groups. In previous work, we had utilised stereophotogrammetry of scanning electron micrographs to measure the depth, volume and other parameters of the individual lacunae. However, increasing experience with the method has suggested that we can await a wide range of biological variability in single cell function in any one experiment. We have therefore tested other methods from which data could be obtained more rapidly to permit a better statistical analysis, albeit with reduced accuracy, of each resorption complex. The main aim of the studies reported here was to evaluate various methods of optical and scanning electron microscopy that can be used for the visualization of osteoclasts and their associated resorption lacunae generated in vitro in slabs of dentine and bone. Optical microscopy was found to be complementary to SEM, enabling vital microscopy of unstained and stained cells. In particular, oblique illumination LM and tandem scanning reflected LM (TSRLM) proved to be of paramount value for this purpose. Fixed coated specimens could be most rapidly scanned for resorption lacunae using darkfield reflected LM or TSRLM.

  20. Cellular resolution ex vivo imaging of gastrointestinal tissues with optical coherence microscopy

    PubMed Central

    Aguirre, Aaron D.; Chen, Yu; Bryan, Bradley; Mashimo, Hiroshi; Huang, Qin; Connolly, James L.; Fujimoto, James G.

    2010-01-01

    Optical coherence microscopy (OCM) combines confocal microscopy and optical coherence tomography (OCT) to improve imaging depth and contrast, enabling cellular imaging in human tissues. We aim to investigate OCM for ex vivo imaging of upper and lower gastrointestinal tract tissues, to establish correlations between OCM imaging and histology, and to provide a baseline for future endoscopic studies. Co-registered OCM and OCT imaging were performed on fresh surgical specimens and endoscopic biopsy specimens, and images were correlated with histology. Imaging was performed at 1.06-μm wavelength with <2-μm transverse and <4-μm axial resolution for OCM, and at 14-μm transverse and <3-μm axial resolution for OCT. Multiple sites on 75 tissue samples from 39 patients were imaged. OCM enabled cellular imaging of specimens from the upper and lower gastrointestinal tracts over a smaller field of view compared to OCT. Squamous cells and their nuclei, goblet cells in Barrett’s esophagus, gastric pits and colonic crypts, and fine structures in adenocarcinomas were visualized. OCT provided complementary information through assessment of tissue architectural features over a larger field of view. OCM may provide a complementary imaging modality to standard OCT approaches for endoscopic microscopy. PMID:20210470

  1. 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.

  2. 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.

  3. Vector near-field calculation of scanning near-field optical microscopy probes using Borgnis potentials as auxiliary functions.

    PubMed

    Wang, Xueen; Fan, Zhaozhong; Tang, Tiantong

    2005-07-01

    A new boundary integral equation method for solving the near field in three-dimensional vector form in scanning near-field optical microscopy (SNOM) using Borgnis potentials as auxiliary functions is presented. A boundary integral equation of the electromagnetic fields, expressed by Borgnis potentials, is derived based on Green's theorem. The harmonic expansion in rotationally symmetric SNOM probe--sample systems is studied, and the three-dimensional electromagnetic problem is partly simplified into a two-dimensional one. The boundary conditions of Borgnis potentials both on dielectric boundaries and on perfectly conducting boundaries are derived. Relevant algorithms were studied, and a computer program was written. As an example, a SNOM probe-sample system composed of a round metal-covered probe and a sample with a flat surface has been numerically studied, and the computational results are given. This new method can be used efficiently for other electromagnetic field problems with round subwavelength structures.

  4. 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.

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

    PubMed Central

    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

  6. 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

  7. 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.

  8. Probing orientation and rotation of red blood cells in optical tweezers by digital holographic microscopy

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

    Interaction of red blood cells (RBC) with optical tweezers has been found to differ under varied physiological and pathological conditions as compared to its normal conditions. Earlier, we reported difference in rotation of trapped RBC in hypertonic conditions for detection of malaria infection. Disk-like RBC when trapped in optical tweezers get oriented in the vertical plane to maximize interaction with trapping beam. However, classical bright field, phase contrast or epifluorescence microscopy cannot confirm its orientation, thus leading to ambiguous conclusions such as folding of RBC during trapping by some researchers. Now, with use of digital holographic microscopy (DHM), we achieved high axial sensitivity that confirmed orientation of trapped red blood cell. Further, DHM enabled quantitative phase imaging of RBC under hypertonic condition. Dynamic changes of rotating RBC under optical tweezers at different trapping laser power were evaluated by the use of DHM. The deviation from linear dependence of rotation speed of RBC on laser power, was attributed towards deformation of RBC shape due to higher laser power (or speed).

  9. 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

    2014-04-30

    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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. Widely tuneable fiber optical parametric amplifier for coherent anti-Stokes Raman scattering microscopy.

    PubMed

    Chemnitz, Mario; Baumgartl, Martin; Meyer, Tobias; Jauregui, Cesar; Dietzek, Benjamin; Popp, Jürgen; Limpert, Jens; Tünnermann, Andreas

    2012-11-19

    We present a narrow-bandwidth, widely tunable fiber laser source for coherent anti-Stokes Raman scattering (CARS) spectro-microscopy. The required, synchronized, two-color pulse trains are generated by optical-parametric amplification in a photonic-crystal fiber (PCF). The four-wave-mixing process in the PCF is pumped by a 140ps, alignment-free fiber laser system, and it is seeded by a tunable continuous-wave laser; hence, a high spectral resolution of up to 1cm(-1) is obtained in the CARS process. Since the PCF is pumped close to its zero-dispersion wavelength, a broad parametric gain can be accessed, resulting in a large tuning range for the generated signal and idler wavelengths. CARS spectroscopy and microscopy is demonstrated, probing different molecular vibrational modes within the accessible region between 1200cm(-1) and 3800cm(-1). PMID:23187513

  18. 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.

  19. 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.

  20. Combining digital holographic microscopy and optical tweezers: a new route in microfluidic

    NASA Astrophysics Data System (ADS)

    Miccio, L.; Memmolo, P.; Merola, F.; Paturzo, M.; Finizio, A.; Grilli, S.; Ferraro, P.

    2012-04-01

    An optical configuration is realized to obtain quantitative phase-contrast maps able to characterize particles floating in a microfluidic chamber by interference microscopy. The novelty is the possibility to drive the sample and measure it thorough the same light path. That is realized by an optical setup made of two light beams coming from the same laser source. One beam provides the optical forces for driving the particle along the desired path and, at same time, it works as object beam in the digital holographic microscope (DHM). The second one acts as reference beam, allowing recording of an interference fringe pattern (i.e., the digital hologram) in an out-of-focus image plane. This work finds application in the field of micromanipulation as, the devise developed allows to operate in microfluidic chambers driving samples flowing in very small volumes. Recently, the field of optical particle micro-manipulation has had rapid growth, due to Optical Tweezers development. A particle is trapped or moved along certain trajectories according to the intensity and phase distribution of the laser beam used. Here, particles freely floating are driven by optical forces along preferential directions and then analyzed by a DHM to numerically calculate their phase-contrast signature. The improvement is that one laser source is employed for making two jobs: driving and analyze the sample. We use two slightly off-axis laser beams coming from a single laser source. The interference between them gives the possibility to record in real-time a sequence of digital holograms, while one of the beam creates the driving force. By this method, a great amount of particles can be analyzed by a real-time recording of DH movies. This allows one to examine each particle at time and characterize it. The optical configuration and the working method are illustrated. Experimental results are shown for polymeric particles and in-vitro.

  1. 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

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

    PubMed

    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.

  3. 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.

  4. 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.

  5. Assessment of colloid response by nonlinear optical microscopy after preoperative radiochemotherapy for rectal carcinoma.

    PubMed

    Li, Lianhuang; Chen, Zhifen; Wang, Xingfu; Zhuo, Shuangmu; Li, Hongsheng; Jiang, Weizhong; Guan, Guoxian; Chen, Jianxin

    2015-05-01

    Colloid response is a type of tumor response that occurs after preoperative radiochemotherapy for rectal carcinoma. Given its important influence on survival, the colloid response should be considered when estimating histopathological reactions. Here, multiphoton microscopy (MPM) was applied to evaluate the colloid response ex vivo. This study demonstrated that MPM has the capability to visualize the colloid response in the absence of labels and can, in particular, identify rare residual carcinomatous cells in mucin pools. These results highlight the potential of this nonlinear optical technique as a diagnostic tool for tumor response after neoadjuvant treatment.

  6. 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.

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

    PubMed

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

    2013-05-20

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

  8. 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.

  9. 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.

  10. Computational signal-to-noise ratio analysis for optical quadrature microscopy.

    PubMed

    Warger, William C; DiMarzio, Charles A

    2009-02-16

    Optical quadrature microscopy (OQM) was invented in 1997 to reconstruct a full-field image of quantitative phase, and has been used to count the number of cells in live mouse embryos. Here we present a thorough SNR analysis that incorporates noise terms for fluctuations in the laser, aberrations within the individual paths of the Mach-Zehnder interferometer, and imperfections within the beamsplitters and CCD cameras to create a model for the resultant phase measurements. The current RMS error of the OQM phase images has been calculated to be 0.08 radians from substituting images from the instrumentation into the model.

  11. 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

  12. Optical-resolution photoacoustic microscopy of angiogenesis in a transgenic mouse model

    NASA Astrophysics Data System (ADS)

    Hu, Song; Oladipupo, Sunday; Yao, Junjie; Santeford, Andrea C.; Maslov, Konstantin; Kovalski, Joanna; Arbeit, Jeffrey M.; Wang, Lihong V.

    2010-02-01

    A major obstacle in studying angiogenesis is the inability to noninvasively image neovascular development in an individual animal. We applied optical-resolution photoacoustic microscopy (OR-PAM) to determine the kinetics of hypoxia-inducible factor-1 (HIF-1)-mediated angiogenesis in a transgenic mouse model. During continuous 30-day activation of HIF-1α, we used OR-PAM to monitor alterations in microvasculature in transgenic mice compared to nontransgenic mice. OR-PAM has demonstrated the potential to precisely monitor antiangiogenic therapy of human cancers, allowing for rapid determinations of therapeutic efficacy or resistance.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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

  18. Optimizing noise for defect analysis with through-focus scanning optical microscopy

    NASA Astrophysics Data System (ADS)

    Attota, Ravikiran; Kramar, John

    2016-03-01

    Through-focus scanning optical microscopy (TSOM) shows promise for patterned defect analysis, but it is important to minimize total system noise. TSOM is a three-dimensional shape metrology method that can achieve sub-nanometer measurement sensitivity by analyzing sets of images acquired through-focus using a conventional optical microscope. Here we present a systematic noise-analysis study for optimizing data collection and data processing parameters for TSOM and then demonstrate how the optimized parameters affect defect analysis. We show that the best balance between signalto- noise performance and acquisition time can be achieved by judicious spatial averaging. Correct background-signal subtraction of the imaging-system inhomogeneities is also critical, as well as careful alignment of the constituent images used in differential TSOM analysis.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. On-chip integrated lensless microscopy module for optical monitoring of adherent growing mammalian cells.

    PubMed

    Li, Wei; Knoll, Thorsten; Thielecke, Hagen

    2010-01-01

    Lab-on-a-chip systems are increasingly applied in cell-based assays for toxicology and drug testing. In this paper, an on-chip integrated lensless microscopy module using a direct projection method for optical monitoring of the shadow images of adherent growing mammalian cells is presented. The biological cells are conserved and interfaced by a microfabricated cavity chip with a 1 microm thick silicon nitride (Si(3)N(4)) substrate onto the surface of a 5 megapixel CMOS image sensor with 2.2 microm pixel size. The optical resolution of the assembly is estimated by the contact/proximate printing theory from optical lithography. Further characterization is made by imaging microbeads in chips with the Si(3)N(4)-membrane as well as in cavity chips with membranes made from dry film resist (DFR, thickness 20, 40 and 60 microm). The module represents a 3 × optical microscope for cell morphology imaging. The function is demonstrated by the growth monitoring of L929 cells cultured in cavity chips with Si(3)N(4) substrate for 2 days and by checking the colorimetric staining of cells with a compromised membrane. PMID:21096993

  6. Thermal and optical characterization of photonic integrated circuits by thermoreflectance microscopy

    NASA Astrophysics Data System (ADS)

    Farzaneh, Maryam; Summers, J. A.; Greenberg, K.; Lueerssen, D.; Ram, Rajeev; Hudgings, Janice

    2010-04-01

    One of the biggest challenges in operation of sub-micrometer sized optoelectronic devices is the generation of excess heat that can create hot spots and affect the device performance. In addition, size reduction and monolithic integration of a large number of optoelectronic devices on a photonic integrated circuit (PIC) restrict direct access to optical signals of each component, which renders the characterization of individual elements difficult. In this talk we report on application of the high resolution, all optical thermal imaging technique of thermoreflectance microscopy in characterization of a PIC comprised of cascaded semiconductor optical amplifiers. A combination of thermal imaging with a comprehensive heat exchange model allows us to obtain internal optical power distribution of the individual devices on a PIC under operating conditions. Other applications of the thermoreflectance technique will also be reviewed. http://meetings.aps.org/link/BAPS.2010.OSS.B1.3

  7. 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

  8. Dynamic wavefront shaping with an acousto-optic lens for laser scanning microscopy.

    PubMed

    Konstantinou, George; Kirkby, Paul A; Evans, Geoffrey J; Naga Srinivas Nadella, K M; Griffiths, Victoria A; Mitchell, John E; Angus Silver, R

    2016-03-21

    Acousto-optic deflectors (AODs) arranged in series and driven with linearly chirped frequencies can rapidly focus and tilt optical wavefronts, enabling high-speed 3D random access microscopy. Non-linearly chirped acoustic drive frequencies can also be used to shape the optical wavefront allowing a range of higher-order aberrations to be generated. However, to date, wavefront shaping with AODs has been achieved by using single laser pulses for strobed illumination to 'freeze' the moving acoustic wavefront, limiting voxel acquisition rates. Here we show that dynamic wavefront shaping can be achieved by applying non-linear drive frequencies to a pair of AODs with counter-propagating acoustic waves, which comprise a cylindrical acousto-optic lens (AOL). Using a cylindrical AOL we demonstrate high-speed continuous axial line scanning and the first experimental AOL-based correction of a cylindrical lens aberration at 30 kHz, accurate to 1/35th of a wave at 800 nm. Furthermore, we develop a model to show how spherical aberration, which is the major aberration in AOL-based remote-focusing systems, can be partially or fully corrected with AOLs consisting of four or six AODs, respectively. PMID:27136821

  9. 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

  10. Multiphoton imaging microscopy at deeper layers with adaptive optics control of spherical aberration.

    PubMed

    Bueno, Juan M; Skorsetz, Martin; Palacios, Raquel; Gualda, Emilio J; Artal, Pablo

    2014-01-01

    Despite the inherent confocality and optical sectioning capabilities of multiphoton microscopy, three-dimensional (3-D) imaging of thick samples is limited by the specimen-induced aberrations. The combination of immersion objectives and sensorless adaptive optics (AO) techniques has been suggested to overcome this difficulty. However, a complex plane-by-plane correction of aberrations is required, and its performance depends on a set of image-based merit functions. We propose here an alternative approach to increase penetration depth in 3-D multiphoton microscopy imaging. It is based on the manipulation of the spherical aberration (SA) of the incident beam with an AO device while performing fast tomographic multiphoton imaging. When inducing SA, the image quality at best focus is reduced; however, better quality images are obtained from deeper planes within the sample. This is a compromise that enables registration of improved 3-D multiphoton images using nonimmersion objectives. Examples on ocular tissues and nonbiological samples providing different types of nonlinear signal are presented. The implementation of this technique in a future clinical instrument might provide a better visualization of corneal structures in living eyes.

  11. 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.

  12. 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

  13. 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

  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. Analysis of anomalous slip in Ta single crystals using optical, atomic force, and orientation imaging microscopies

    SciTech Connect

    Stoelken, J.S.; King, W.E.; Schwartz, A.J.; Campbell, G.H.; Balooch, M.

    1999-07-01

    High purity Ta single crystals oriented for single slip were deformed in compression at 300K and 77K. The sample deformed at 300K exhibited wavy glide whereas the sample deformed at 77K exhibited anomalous slip. Sharp load drops were recorded in the stress-strain curve of the sample tested at 77K. Previous work attributes such unloading events to either the formation of large deformation twins or to the anomalous slip process itself. Orientation imaging microscopy was applied to probe lattice rotations occurring as a result of deformation in an effort to detect the presence of large deformation twins, none were found. Optical and atomic force microscopies were applied to map the slip traces appearing on the sample surface. Atomic force microscopy revealed that the fine structure within the rather coarse anomalous slip bands is comprised of atomistic scale slip lines organized into packets. These slip packets appear to account for the fine slip traces often observed within anomalous slip bands.

  16. 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.

  17. Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy

    PubMed Central

    Young, Jonathan W; Locke, James C W; Altinok, Alphan; Rosenfeld, Nitzan; Bacarian, Tigran; Swain, Peter S; Mjolsness, Eric; Elowitz, Michael B

    2014-01-01

    Quantitative single-cell time-lapse microscopy is a powerful method for analyzing gene circuit dynamics and heterogeneous cell behavior. We describe the application of this method to imaging bacteria by using an automated microscopy system. This protocol has been used to analyze sporulation and competence differentiation in Bacillus subtilis, and to quantify gene regulation and its fluctuations in individual Escherichia coli cells. The protocol involves seeding and growing bacteria on small agarose pads and imaging the resulting microcolonies. Images are then reviewed and analyzed using our laboratory's custom MATLAB analysis code, which segments and tracks cells in a frame-to-frame method. This process yields quantitative expression data on cell lineages, which can illustrate dynamic expression profiles and facilitate mathematical models of gene circuits. With fast-growing bacteria, such as E. coli or B. subtilis, image acquisition can be completed in 1 d, with an additional 1–2 d for progressing through the analysis procedure. PMID:22179594

  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

    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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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

  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. Optically stabilized mercury short-arc lamp as UV light source for microscopy

    NASA Astrophysics Data System (ADS)

    Heynen, Susanne; Gough, David A.; Price, Jeffrey H.

    1997-05-01

    For certain applications in microscopy, mercury vapor short arc lamps are utilized as UV-light sources because of their high intensity and their road spectrum. Unfortunately, they are also very unstable. Especially for single wavelength fluorescence image cytometry, there is a need for a stable, high intensity light source. Substantially improved stability was achieved using optical feedback and fiberoptic scrambling. The system uses a photodiode to monitor the light intensity, and feeds the readout back to a controller. The controller compares this readout to a preset reference voltage and adjusts the lamp supply current accordingly. The optical fiber scrambles the light to correct the effects of arc wander. Preliminary results of performance tests of this system show a coefficient of variation (CV) of less than 0.1 percent over 20 hours at a sample frequency of 30 Hz. This CV is a factor of 30 better than a conventional current stabilized mercy vapor short arc lamp. Scrambled optical feedback is a necessary addition for systems with mercury short arc lamps, especially for image fluorometry applications.

  8. 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

  9. 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

  10. Three dimensional optical manipulation and structural imaging of soft materials by use of laser tweezers and multimodal nonlinear microscopy.

    PubMed

    Trivedi, Rahul P; Lee, Taewoo; Bertness, Kris A; Smalyukh, Ivan I

    2010-12-20

    We develop an integrated system of holographic optical trapping and multimodal nonlinear microscopy and perform simultaneous three-dimensional optical manipulation and non-invasive structural imaging of composite soft-matter systems. We combine different nonlinear microscopy techniques such as coherent anti-Stokes Raman scattering, multi-photon excitation fluorescence and multi-harmonic generation, and use them for visualization of long-range molecular order in soft materials by means of their polarized excitation and detection. The combined system enables us to accomplish manipulation in composite soft materials such as colloidal inclusions in liquid crystals as well as imaging of each separate constituents of the composite material in different nonlinear optical modalities. We also demonstrate optical generation and control of topological defects and simultaneous reconstruction of their three-dimensional long-range molecular orientational patterns from the nonlinear optical images.

  11. Computational-optical microscopy for 3D biological imaging beyond the diffraction limit

    NASA Astrophysics Data System (ADS)

    Grover, Ginni

    In recent years, super-resolution imaging has become an important fluorescent microscopy tool. It has enabled imaging of structures smaller than the optical diffraction limit with resolution less than 50 nm. Extension to high-resolution volume imaging has been achieved by integration with various optical techniques. In this thesis, development of a fluorescent microscope to enable high resolution, extended depth, three dimensional (3D) imaging is discussed; which is achieved by integration of computational methods with optical systems. In the first part of the thesis, point spread function (PSF) engineering for volume imaging is discussed. A class of PSFs, referred to as double-helix (DH) PSFs, is generated. The PSFs exhibit two focused spots in the image plane which rotate about the optical axis, encoding depth in rotation of the image. These PSFs extend the depth-of-field up to a factor of ˜5. Precision performance of the DH-PSFs, based on an information theoretical analysis, is compared with other 3D methods with conclusion that the DH-PSFs provide the best precision and the longest depth-of-field. Out of various possible DH-PSFs, a suitable PSF is obtained for super-resolution microscopy. The DH-PSFs are implemented in imaging systems, such as a microscope, with a special phase modulation at the pupil plane. Surface-relief elements which are polarization-insensitive and ˜90% light efficient are developed for phase modulation. The photon-efficient DH-PSF microscopes thus developed are used, along with optimal position estimation algorithms, for tracking and super-resolution imaging in 3D. Imaging at depths-of-field of up to 2.5 microm is achieved without focus scanning. Microtubules were imaged with 3D resolution of (6, 9, 39) nm, which is in close agreement with the theoretical limit. A quantitative study of co-localization of two proteins in volume was conducted in live bacteria. In the last part of the thesis practical aspects of the DH-PSF microscope are

  12. 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

  13. Multimodal In Vivo Skin Imaging with Integrated Optical Coherence and Multiphoton Microscopy

    PubMed Central

    Graf, Benedikt W.; Boppart, Stephen A.

    2014-01-01

    In this paper, we demonstrate high-resolution, multimodal in vivo imaging of human skin using optical coherence (OCM) and multiphoton microscopy (MPM). These two modalities are integrated into a single instrument to enable simultaneous acquisition and coregistration. The system design and the OCM image processing architecture enable sufficient performance of both modalities for in vivo imaging of human skin. Examples of multimodal in vivo imaging are presented as well as time lapse imaging of blood flow in single capillary loops. By making use of multiple intrinsic contrast mechanisms this integrated technique improves the ability to noninvasively visualize living tissue. Integrated OCM and MPM has potential applications for in vivo diagnosis of various pathological skin conditions, such as skin cancer, as well as potential pharmaceutical and cosmetic research applications. PMID:25673966

  14. 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

  15. 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.

  16. Optical-Resolution Photoacoustic Microscopy for Volumetric and Spectral Analysis of Histological and Immunochemical Samples**

    PubMed Central

    Zhang, Yu Shrike; Yao, Junjie; Zhang, Chi; Li, Lei; Wang, Lihong V.; Xia, Younan

    2014-01-01

    Optical-resolution photoacoustic microscopy (OR-PAM) is an imaging modality with superb penetration depth and excellent absorption contrast. Here we demonstrate, for the first time, that this technique can advance quantitative analysis of conventional chromogenic histochemistry. Because OR-PAM can quantify the absorption contrast at different wavelengths, it is feasible to spectrally resolve the specific biomolecules involved in a staining color. Furthermore, the tomographic capability of OR-PAM allows for non-invasive volumetric imaging of a thick sample without microtoming it. By immunostaining the sample with different chromogenic agents, we further demonstrated the ability of OP-PAM to resolve different types of cells in a co-culture sample with imaging depths up to 1 mm. Taken together, the integration of OR-PAM with (immuno)histochemistry offers a simple and versatile technique with broad applications in cell biology, pathology, tissue engineering, and related biomedical studies. PMID:24961608

  17. Crystallization kinetics of poly-(lactic acid) with and without talc: Optical microscopy and calorimetric analysis

    NASA Astrophysics Data System (ADS)

    Refaa, Z.; Boutaous, M.; Rousset, F.; Fulchiron, R.; Zinet, M.; Xin, S.; Bourgin, P.

    2014-05-01

    Poly-(lactic acid) or PLA is a biodegradable polymer synthesized from renewable resources. Recently, the discovery of new polymerization routes has allowed increasing the produced volumes. As a consequence, PLA is becoming of great interest for reducing the dependence on petroleum-based plastics. Because of its interesting mechanical properties, PLA is seen as a potential substitute for some usual polymers. However, its relatively slow crystallization kinetics can be a disadvantage with regard to industrial applications. The crystallization kinetics of PLA can be enhanced by adding nucleating agents, which also influences on crystalline morphology and rheological behavior. In the present work, the isothermal quiescent crystallization kinetics of both neat PLA and PLA/talc composite (5 wt% talc) are investigated. The effects of talc on the overall crystallization kinetics and on the crystalline morphology are analyzed using both optical microscopy measurements and thermal analysis by differential scanning calorimetry.

  18. 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.

  19. 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.

  20. Characterization of the polycaprolactone melt crystallization: complementary optical microscopy, DSC, and AFM studies.

    PubMed

    Speranza, V; Sorrentino, A; De Santis, F; Pantani, R

    2014-01-01

    The first stages of the crystallization of polycaprolactone (PCL) were studied using several techniques. The crystallization exotherms measured by differential scanning calorimetry (DSC) were analyzed and compared with results obtained by polarized optical microscopy (POM), rheology, and atomic force microscope (AFM). The experimental results suggest a strong influence of the observation scale. In particular, the AFM, even if limited on time scale, appears to be the most sensitive technique to detect the first stages of crystallization. On the contrary, at least in the case analysed in this work, rheology appears to be the least sensitive technique. DSC and POM provide closer results. This suggests that the definition of induction time in the polymer crystallization is a vague concept that, in any case, requires the definition of the technique used for its characterization.

  1. 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.

  2. 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

  3. Use of Confocal Microscopy in the Search for Optical Counterparts to Gamma-Ray Bursts

    NASA Astrophysics Data System (ADS)

    Sharma, A.; Jacobson, R. E.

    1995-09-01

    A number of star-like images have been found on archival photographic plates, however there is debate as to whether these are optical emissions relating to Gamma-Ray bursts, or simply plate defects. Results from photographic science are summarized so that the characteristic shape of different types of images on photographic plates can be theoretically predicted. Explanation for the formation of the relief image and reasons for its continued study are described. New methods in the use of laser scanning confocal microscopy are presented to study the relief image and the depthwise disposition of silver so that a test procedure can be established to end the speculation surrounding the astrophysical truth of stellar-like images on photographic plates.

  4. 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.

  5. Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart.

    PubMed

    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.

  6. 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

  7. 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

  8. Intensity Fluctuations of Optical Microscopy as a Means to Measure Axial Diffusion

    NASA Astrophysics Data System (ADS)

    Bihari, Malvika; Russell, Thomas; Hoagland, David

    2009-03-01

    Via optical microscopy, geometrically hindered motions of a single large solute (particle or polymer) can be imaged in real time. Here, intensity fluctuations of confocal fluorescence microscopy admit another way to probe such motions, one convenient when motions are perpendicular to a planar substrate. The focal plane is positioned within the substrate (lying on the microscope stage) and intensity fluctuations arise from motions in-and out- of the focal volume. Two experiments illustrate the new approach, diffusion within pores of a planar membrane or in solution near a solid wall. In the first, diffusion coefficients of spherical particles were measured inside pores of a track-etched polycarbonate membrane as functions of particle and pore size. In the second, anisotropic diffusion (perpendicular/parallel) of the same particles was measured within a few particle diameters of a solid boundary. Theory for hydrodynamically hindered diffusion in both cases is well developed, and data are compared to predictions. Two ways to assess particle/polymer motion, tracking single particles and correlating intensity fluctuations, will be discussed.

  9. 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

    2016-05-19

    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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. Preparation of mica supported lipid bilayers for high resolution optical microscopy imaging.

    PubMed

    Matysik, Artur; Kraut, Rachel S

    2014-06-07

    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.

  18. 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

  19. 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.

  20. Combined spectrally encoded confocal microscopy and optical frequency domain imaging system

    NASA Astrophysics Data System (ADS)

    Kang, DongKyun; Suter, Melissa J.; Boudoux, Caroline; Yachimski, Patrick S.; Bouma, Brett E.; Nishioka, Norman S.; Tearney, Guillermo J.

    2009-02-01

    Spectrally encoded confocal microscopy (SECM) and optical frequency domain imaging (OFDI) are two reflectancebased imaging technologies that may be utilized for high-resolution microscopic screening of internal organs. SECM provides en face images of tissues with a high lateral resolution of 1-2 μm, and a penetration depth of up to 300 μm. OFDI generates cross-sectional images of tissue architecture with a resolution of 10-20 μm and a penetration depth of 1- 2 mm. Since the two technologies yield complementary microscopic information on two different size scales (SECM-cellular and OFDI-architectural) that are commonly used for histopathologic evaluation, their combination may allow for more accurate optical diagnosis. Here, we report the integration of these two imaging modalities in a single bench top system. SECM images of swine small intestine showed the presence of goblet cells, and OFDI images revealed the finger-shaped villous architecture. In clinical study of 9 gastroesophageal biopsies from 8 patients, a diverse set of architectural and cellular features was observed, including squamous mucosa with mild hyperplasia and gastric antral mucosa with gastric pits and crypts. The capability of this multimodality device to enable the visualization of microscopic features on these two size scales supports our hypothesis that improved diagnostic accuracy may be obtained by merging these two technologies into a single instrument.

  1. Fast optical-resolution photoacoustic microscopy using a 2-axis water-proofing MEMS scanner.

    PubMed

    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 mm(2) 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 mm(3). 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.

  2. 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.

  3. 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.

  4. In vivo visualization of skin inflammation by optical coherence tomography and two-photon microscopy

    PubMed Central

    Kim, Bumju; Lee, Seung Hun; Yoon, Calvin J.; Gho, Yong Song; Ahn, G-One; Kim, Ki Hean

    2015-01-01

    Inflammation is a non-specific immune response to injury intended to protect biological tissue from harmful stimuli such as pathogens, irritants, and damaged cells. In vivo optical tissue imaging has been used to provide spatial and dynamic characteristics of inflammation within the tissue. In this paper, we report in vivo visualization of inflammation in the skin at both cellular and physiological levels by using a combination of label-free two-photon microscopy (TPM) and optical coherence tomography (OCT). Skin inflammation was induced by topically applying lipopolysaccharide (LPS) on the mouse ear. Temporal OCT imaging visualized tissue swelling, vasodilation, and increased capillary density 30 min and 1 hour after application. TPM imaging showed immune cell migration within the inflamed skin. Combined OCT and TPM was applied to obtain complementary information from each modality in the same region of interest. The information provided by each modality were consistent with previous reports about the characteristics of inflammation. Therefore, the combination of OCT and TPM holds potential for studying inflammation of the skin. PMID:26203377

  5. Microstructural characterization of myocardial infarction with optical coherence tractography and two-photon microscopy.

    PubMed

    Goergen, Craig J; Chen, Howard H; Sakadžić, Sava; Srinivasan, Vivek J; Sosnovik, David E

    2016-09-01

    Myocardial infarction leads to complex changes in the fiber architecture of the heart. Here, we present a novel optical approach to characterize these changes in intact hearts in three dimensions. Optical coherence tomography (OCT) was used to derive a depth-resolved field of orientation on which tractography was performed. Tractography of healthy myocardium revealed a smooth linear transition in fiber inclination or helix angle from the epicardium to endocardium. Conversely, in infarcted hearts, no coherent microstructure could be identified in the infarct with OCT Additional characterization of the infarct was performed by the measurement of light attenuation and with two-photon microscopy. Myofibers were imaged using autofluorescence and collagen fibers using second harmonic generation. This revealed the presence of two distinct microstructural patterns in areas of the infarct with high light attenuation. In the presence of residual myofibers, the surrounding collagen fibers were aligned in a coherent manner parallel to the myofibers. In the absence of residual myofibers, the collagen fibers were randomly oriented and lacked any microstructural coherence. The presence of residual myofibers thus exerts a profound effect on the microstructural properties of the infarct scar and consequently the risk of aneurysm formation and arrhythmias. Catheter-based approaches to segment and image myocardial microstructure in humans are feasible and could play a valuable role in guiding the development of strategies to improve infarct healing. PMID:27650248

  6. 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.

  7. Adhesive improvement in optical coherence tomography combined with confocal microscopy for class V cavities investigations

    NASA Astrophysics Data System (ADS)

    Rominu, Mihai; Sinescu, Cosmin; Negrutiu, Meda L.; Rominu, Roxana O.; Pop, Daniela M.; Topala, Florin; Stoia, Adelina; Petrescu, Emanuela; Bradu, Adrian; Dobre, George; Podoleanu, Adrian G.

    2010-03-01

    The purpose of this study is to present a non invasive method for the marginal adaptation evaluation in class V composite restorations. Standardized class V cavities prepared in human extracted teeth were filled with composite resin (Premise, Kerr). The specimens were thermocycled. The interfaces were examined by Optical Coherence Tomography (OCT) combined with confocal microscopy and fluorescence. The optical configuration uses two single mode directional couplers with a superluminiscent diode as the source at 1300 nm. The scanning procedure is similar to that used in any confocal microscope, where the fast scanning is en-face (line rate) and the depth scanning is much slower (at the frame rate). Gaps at the interfaces as well as on the inside of the composite resin were identified. OCT has numerous advantages that justify its in vivo and in vitro use compared to conventional techniques. One of the main concerns was the fact that at the adhesive layer site it was very hard to tell the adhesive apart from material defects. For this reason the adhesive was optimized in order to be more scattering. This way we could make a difference between the adhesive layer and the material defects that could lead to microleakages.

  8. 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.

  9. Fluorescent nanoscale detection of biotin streptavidin interaction using near-field scanning optical microscopy

    NASA Astrophysics Data System (ADS)

    Park, Hyun Kyu; Gokarna, Anisha; Hulme, John P.; Park, Hyun Gyu; Chung, Bong Hyun

    2008-06-01

    We describe a nanoscale strategy for detecting biotin-streptavidin binding using near-field scanning optical microscopy (NSOM) that exploits the fluorescence properties of single polydiacetylene (PDA) liposomes. NSOM is more useful to observe nanomaterials having optical properties with the help of topological information. We synthesized amine-terminated 10,12-pentacosadiynoic acid (PCDA) monomer (PCDA-NH2) and used this derivatized monomer to prepare PCDA liposomes. PCDA-NH2 liposomes were immobilized on an aldehyde-functionalized glass surface followed by photopolymerization by using a 254 nm light source. To measure the biotin-streptavidin binding, we conjugated photoactivatable biotin to immobilized PCDA-NH2 liposomes by UV irradiation (365 nm) and subsequently allowed them to interact with streptavidin. We analyzed the fluorescence using a fluorescence scanner and observed single liposomes using NSOM. The average height and NSOM signal observed in a single liposome after binding were ~31.3 to 8.5 ± 0.5 nm and 0.37 to 0.16 ± 0.6 kHz, respectively. This approach, which has the advantage of not requiring a fluorescent label, could prove highly beneficial for single molecule detection technology.

  10. Long-term in vivo study of vertebrate embryonic development using noninvasive harmonics optical microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Szu-Yu; Hsieh, C.-S.; Chu, S.-W.; Lin, Cheng-Yung; Ko, C.-Y.; Chen, Y.-C.; Tsai, Huai-Jen; Hu, C.-H.; Sun, Chi-Kuang

    2005-03-01

    Harmonics optical microscopy (HOM) provides a truly "noninvasive" tool for in vivo and long-term study of vertebrate embryonic development. Based on the nonlinear natures, it provides sub-micrometer 3D spatial resolution and high 3D optical-sectioning power (~1μm axial resolution) without using invasive and toxic fluorophores. Since only virtual-level-transition is involved, HOM is known to leave no energy deposition and no photodamages. Combined with second harmonic generation, which is sensitive to specific structure such as nerve and muscle fibers, HOM can be used to do functional studies of early developmental dynamics of many vertebrate physiological systems. Recently, zebrafish has become a standard model for many biological and medical studies of vertebrates, due to the similarity between embryonic development of zebrafish and human being. Zebrafish embryos now have been used to study many vertebrate physiological systems. We have demonstrated an in vivo HOM study of developmental dynamics of several embryonic physiological systems in live zebrafish embryos, with focuses on the developments of brains, eyes, ears, and hearts. Based on a femtosecond Cr:forsterite laser, which provides the deepest penetration (~1.5mm) and least photodamage in the zebrafish embryo, complete developing processes of different physiological systems within a period of time longer than 20 hours can be non-invasively observed inside the same embryo.

  11. Depth-Encoded Spectral Domain Phase Microscopy for Simultaneous Multi-Site Nanoscale Optical Measurements

    PubMed Central

    Hendargo, Hansford C.; Bower, Bradley A.; Reinstein, Alex S.; Shepherd, Neal; Tao, Yuankai K.; Izatt, Joseph A.

    2011-01-01

    Spectral domain phase microscopy (SDPM) is an extension of spectral domain optical coherence tomography (SDOCT) that exploits the extraordinary phase stability of spectrometer-based systems with common-path geometry to resolve sub-wavelength displacements within a sample volume. This technique has been implemented for high resolution axial displacement and velocity measurements in biological samples, but since axial displacement information is acquired serially along the lateral dimension, it has been unable to measure fast temporal dynamics in extended samples. Depth-Encoded SDPM (DESDPM) uses multiple sample arms with unevenly spaced common path reference reflectors to multiplex independent SDPM signals from separate lateral positions on a sample simultaneously using a single interferometer, thereby reducing the time required to detect unique optical events to the integration period of the detector. Here, we introduce DESDPM and demonstrate the ability to acquire useful phase data concurrently at two laterally separated locations in a phantom sample as well as a biological preparation of spontaneously beating chick cardiomyocytes. DESDPM may be a useful tool for imaging fast cellular phenomena such as nervous conduction velocity or contractile motion. PMID:21886940

  12. Depth-Encoded Spectral Domain Phase Microscopy for Simultaneous Multi-Site Nanoscale Optical Measurements.

    PubMed

    Hendargo, Hansford C; Bower, Bradley A; Reinstein, Alex S; Shepherd, Neal; Tao, Yuankai K; Izatt, Joseph A

    2011-09-01

    Spectral domain phase microscopy (SDPM) is an extension of spectral domain optical coherence tomography (SDOCT) that exploits the extraordinary phase stability of spectrometer-based systems with common-path geometry to resolve sub-wavelength displacements within a sample volume. This technique has been implemented for high resolution axial displacement and velocity measurements in biological samples, but since axial displacement information is acquired serially along the lateral dimension, it has been unable to measure fast temporal dynamics in extended samples. Depth-Encoded SDPM (DESDPM) uses multiple sample arms with unevenly spaced common path reference reflectors to multiplex independent SDPM signals from separate lateral positions on a sample simultaneously using a single interferometer, thereby reducing the time required to detect unique optical events to the integration period of the detector. Here, we introduce DESDPM and demonstrate the ability to acquire useful phase data concurrently at two laterally separated locations in a phantom sample as well as a biological preparation of spontaneously beating chick cardiomyocytes. DESDPM may be a useful tool for imaging fast cellular phenomena such as nervous conduction velocity or contractile motion.

  13. 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

  14. 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.

  15. 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.

  16. Refractometry of melanocyte cell nuclei using optical scatter images recorded by digital Fourier microscopy

    NASA Astrophysics Data System (ADS)

    Seet, Katrina Y. T.; Nieminen, Timo A.; Zvyagin, Andrei V.

    2009-07-01

    The cell nucleus is the dominant optical scatterer in the cell. Neoplastic cells are characterized by cell nucleus polymorphism and polychromism-i.e., the nuclei exhibits an increase in the distribution of both size and refractive index. The relative size parameter, and its distribution, is proportional to the product of the nucleus size and its relative refractive index and is a useful discriminant between normal and abnormal (cancerous) cells. We demonstrate a recently introduced holographic technique, digital Fourier microscopy (DFM), to provide a sensitive measure of this relative size parameter. Fourier holograms were recorded and optical scatter of individual scatterers were extracted and modeled with Mie theory to determine the relative size parameter. The relative size parameter of individual melanocyte cell nuclei were found to be 16.5+/-0.2, which gives a cell nucleus refractive index of 1.38+/-0.01 and is in good agreement with previously reported data. The relative size parameters of individual malignant melanocyte cell nuclei are expected to be greater than 16.5.

  17. 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.

  18. 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.

  19. Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors.

    PubMed

    Salomé, R; Kremer, Y; Dieudonné, S; Léger, J-F; Krichevsky, O; Wyart, C; Chatenay, D; Bourdieu, L

    2006-06-30

    Two-photon scanning microscopy (TPSM) is a powerful tool for imaging deep inside living tissues with sub-cellular resolution. The temporal resolution of TPSM is however strongly limited by the galvanometric mirrors used to steer the laser beam. Fast physiological events can therefore only be followed by scanning repeatedly a single line within the field of view. Because acousto-optic deflectors (AODs) are non-mechanical devices, they allow access at any point within the field of view on a microsecond time scale and are therefore excellent candidates to improve the temporal resolution of TPSM. However, the use of AOD-based scanners with femtosecond pulses raises several technical difficulties. In this paper, we describe an all-digital TPSM setup based on two crossed AODs. It includes in particular an acousto-optic modulator (AOM) placed at 45 degrees with respect to the AODs to pre-compensate for the large spatial distortions of femtosecond pulses occurring in the AODs, in order to optimize the spatial resolution and the fluorescence excitation. Our setup allows recording from freely selectable point-of-interest at high speed (1kHz). By maximizing the time spent on points of interest, random-access TPSM (RA-TPSM) constitutes a promising method for multiunit recordings with millisecond resolution in biological tissues.

  20. 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.

  1. Single myelin fiber imaging in living rodents without labeling by deep optical coherence microscopy.

    PubMed

    Ben Arous, Juliette; Binding, Jonas; Léger, Jean-François; Casado, Mariano; Topilko, Piotr; Gigan, Sylvain; Boccara, A Claude; 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.

  2. 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.

  3. Energy Landscape of Alginate-Epimerase Interactions Assessed by Optical Tweezers and Atomic Force Microscopy.

    PubMed

    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

  4. 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

  5. Three-dimensional imaging of normal skin and nonmelanoma skin cancer with cellular resolution using Gabor domain optical coherence microscopy.

    PubMed

    Lee, Kye-Sung; Zhao, Huimin; Ibrahim, Sherrif F; Meemon, Natthani; Khoudeir, Laura; Rolland, Jannick P

    2012-12-01

    We investigate morphological differences in three-dimensional (3-D) images with cellular resolution between nonmelanoma skin cancer and normal skin using Gabor domain optical coherence microscopy. As a result, we show for the first time cellular optical coherence images of 3-D features differentiating cancerous skin from normal skin. In addition, in vivo volumetric images of normal skin from different anatomic locations are shown and compared.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. Spatiotemporal control of femtosecond plasmon using plasmon response functions measured by near-field scanning optical microscopy (NSOM).

    PubMed

    Onishi, Shutaro; Matsuishi, Keiichiro; Oi, Jun; Harada, Takuya; Kusaba, Miyuki; Hirosawa, Kenichi; Kannari, Fumihiko

    2013-11-01

    Spectral interferometry combined with near-field scanning optical microscopy is applied in the spatiotemporal characterization of femtosecond plasmon localized at gold nanostructures and surface plasmon polariton in an air-gap waveguide. Based on the plasmon response function in both the amplitude and the phase obtained from the measurements, we deterministically tailored the femtosecond plasmon pulse by shaping the femtosecond excitation laser pulses.

  11. Monitoring the Oxygen Dynamics of Brain Tissue In Vivo by Fast Acousto-Optic Scanning Microscopy: A Proposed Instrument.

    PubMed

    Zhou, Zhenqiao; Chen, Dayu; Huang, Zhiqiang; Wang, Shaofang; Zeng, Shaoqun

    2016-01-01

    The function of the brain neural circuit is highly dependent on oxygen supply. Imaging the precise oxygen distribution and dynamics are critical for understanding the relationship between neuronal activity and oxygen dynamics of the nearby capillaries. Here, we develop fast acousto-optic scanning two-photon microscopy. Combined with oxygen probes, such as PtP-C343, we can monitor oxygen dynamics at the submicron level by this real-time microscopy. In this fast acousto-optic scanning microscopy, an acousto-optic deflector (AOD), an inertia-less scanner, is used to scan the femtosecond laser. A cylindrical lens is used to compensate the 'cylindrical lens effect' of AOD and a prism is used to compensate the chromatic dispersion of AOD. An electro-optical modulator (EOM) and a sCMOS camera are gated to measure the phosphorescence lifetime. With a 40× water objective lens, this set-up can image a 100 μm × 100 μm field of view at a speed of 20 frames per second and a 25 μm × 8 μm field of view at a speed of 500 frames per second. This real-time two-photon microscopy is expected to be a good tool for observing and recording the precise rapid oxygen dynamics in the cerebral cortex, which will facilitate studies of oxygen metabolism in neurosciences. PMID:27526168

  12. 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.

  13. Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies.

    PubMed

    Yang, R; Elankumaran, Y; Hijjawi, N; Ryan, U

    2015-06-01

    A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages.

  14. 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.

  15. 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.

  16. 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.

  17. High-Contrast Fluorescence Microscopy for a Biomolecular Analysis Based on Polarization Techniques Using an Optical Interference Mirror Slide

    PubMed Central

    Yasuda, Mitsuru; Akimoto, Takuo

    2014-01-01

    Fluorescence microscopy with an improved contrast for fluorescence images is developed using an optical interference mirror (OIM) slide, which can enhance the fluorescence from a fluorophore as a result of the double interference of the excitation light and emission light. To improve the contrast of a fluorescence image using an OIM slide, a linearly-polarized excitation light was employed, and the fluorescence emission polarized perpendicular to the polarization of the excitation light was detected. The image contrast with this optical system was improved 110-fold for rhodamine B spotted on the OIM, in comparison with a glass slide using a general fluorescence microscopy optical system. Moreover, a 24-fold improvement of the image contrast was achieved for the detection of Cy3-labeled streptavidin bound to immobilize biotin. PMID:25587437

  18. 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.

  19. 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.

  20. 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.

  1. Swept source optical coherence microscopy using a 1310 nm VCSEL light source

    PubMed Central

    Ahsen, Osman O.; Tao, Yuankai K.; Potsaid, Benjamin M.; Sheikine, Yuri; Jiang, James; Grulkowski, Ireneusz; Tsai, Tsung-Han; Jayaraman, Vijaysekhar; Kraus, Martin F.; Connolly, James L.; Hornegger, Joachim; Cable, Alex; Fujimoto, James G.

    2013-01-01

    We demonstrate high speed, swept source optical coherence microscopy (OCM) using a MEMS tunable vertical cavity surface-emitting laser (VCSEL) light source. The light source had a sweep rate of 280 kHz, providing a bidirectional axial scan rate of 560 kHz. The sweep bandwidth was 117 nm centered at 1310 nm, corresponding to an axial resolution of 13.1 µm in air, corresponding to 8.1 µm (9.6 µm spectrally shaped) in tissue. Dispersion mismatch from different objectives was compensated numerically, enabling magnification and field of view to be easily changed. OCM images were acquired with transverse resolutions between 0.86 µm - 3.42 µm using interchangeable 40X, 20X and 10X objectives with ~600 µm x 600 µm, ~1 mm x 1 mm and ~2 mm x 2 mm field-of-view (FOV), respectively. Parasitic variations in path length with beam scanning were corrected numerically. These features enable swept source OCM to be integrated with a wide range of existing scanning microscopes. Large FOV mosaics were generated by serially acquiring adjacent overlapping microscopic fields and combining them in post-processing. Fresh human colon, thyroid and kidney specimens were imaged ex vivo and compared to matching histology sections, demonstrating the ability of OCM to image tissue specimens. PMID:23938673

  2. 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.

  3. Towards automated segmentation of cells and cell nuclei in nonlinear optical microscopy.

    PubMed

    Medyukhina, Anna; Meyer, Tobias; Schmitt, Michael; Romeike, Bernd F M; Dietzek, Benjamin; Popp, Jürgen

    2012-11-01

    Nonlinear optical (NLO) imaging techniques based e.g. on coherent anti-Stokes Raman scattering (CARS) or two photon excited fluorescence (TPEF) show great potential for biomedical imaging. In order to facilitate the diagnostic process based on NLO imaging, there is need for an automated calculation of quantitative values such as cell density, nucleus-to-cytoplasm ratio, average nuclear size. Extraction of these parameters is helpful for the histological assessment in general and specifically e.g. for the determination of tumor grades. This requires an accurate image segmentation and detection of locations and boundaries of cells and nuclei. Here we present an image processing approach for the detection of nuclei and cells in co-registered TPEF and CARS images. The algorithm developed utilizes the gray-scale information for the detection of the nuclei locations and the gradient information for the delineation of the nuclear and cellular boundaries. The approach reported is capable for an automated segmentation of cells and nuclei in multimodal TPEF-CARS images of human brain tumor samples. The results are important for the development of NLO microscopy into a clinically relevant diagnostic tool.

  4. 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

  5. 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.

  6. 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

  7. 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.

  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. 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.

  10. 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.

  11. 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.

  12. Towards automated segmentation of cells and cell nuclei in nonlinear optical microscopy.

    PubMed

    Medyukhina, Anna; Meyer, Tobias; Schmitt, Michael; Romeike, Bernd F M; Dietzek, Benjamin; Popp, Jürgen

    2012-11-01

    Nonlinear optical (NLO) imaging techniques based e.g. on coherent anti-Stokes Raman scattering (CARS) or two photon excited fluorescence (TPEF) show great potential for biomedical imaging. In order to facilitate the diagnostic process based on NLO imaging, there is need for an automated calculation of quantitative values such as cell density, nucleus-to-cytoplasm ratio, average nuclear size. Extraction of these parameters is helpful for the histological assessment in general and specifically e.g. for the determination of tumor grades. This requires an accurate image segmentation and detection of locations and boundaries of cells and nuclei. Here we present an image processing approach for the detection of nuclei and cells in co-registered TPEF and CARS images. The algorithm developed utilizes the gray-scale information for the detection of the nuclei locations and the gradient information for the delineation of the nuclear and cellular boundaries. The approach reported is capable for an automated segmentation of cells and nuclei in multimodal TPEF-CARS images of human brain tumor samples. The results are important for the development of NLO microscopy into a clinically relevant diagnostic tool. PMID:22811013

  13. Does optical microangiography provide accurate imaging of capillary vessels?: validation using multiphoton microscopy

    PubMed Central

    Wang, Hequn; Baran, Utku; Li, Yuandong; Qin, Wan; Wang, Wenbo; Zeng, Haishan; Wang, Ruikang K.

    2014-01-01

    Abstract. Optical microangiography (OMAG) has been extensively utilized to study three-dimensional tissue vasculature in vivo. However, with the limited image resolution (∼10  μm) of the commonly used systems, some concerns were raised: (1) whether OMAG is capable of providing the imaging of capillary vessels that are of an average diameter of ∼6  μm; (2) if yes, whether OMAG can provide meaningful quantification of vascular density within the scanned tissue volume. Multiphoton microscopy (MPM) is capable of depth-resolved high-resolution (∼1  μm) imaging of biological tissue structures. With externally labeled plasma, the vascular network including single capillaries can be well visualized. We compare the vascular images of in vivo mouse brain acquired by both OMAG and MPM systems. We found that within the penetration depth range of the MPM system, OMAG is able to accurately visualize blood vessels including capillaries. Although the resolution of OMAG may not be able to 100% resolve two closely packed tiny capillaries in tissue, it is still capable of visualizing most of the capillaries because there are interstitial tissue spaces between them. We believe our validation results reinforce the application of OMAG in microvasculature-related studies. PMID:25341071

  14. 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.

  15. 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.

  16. 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.

  17. 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

    2016-10-01

    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.

  18. 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

  19. Automated centreline extraction of neuronal dendrite from optical microscopy image stacks

    NASA Astrophysics Data System (ADS)

    Xiao, Liang; Zhang, Fanbiao

    2010-11-01

    In this work we present a novel vision-based pipeline for automated skeleton detection and centreline extraction of neuronal dendrite from optical microscopy image stacks. The proposed pipeline is an integrated solution that merges image stacks pre-processing, the seed points detection, ridge traversal procedure, minimum spanning tree optimization and tree trimming into to a unified framework to deal with the challenge problem. In image stacks preprocessing, we first apply a curvelet transform based shrinkage and cycle spinning technique to remove the noise. This is followed by the adaptive threshold method to compute the result of neuronal object segmentation, and the 3D distance transformation is performed to get the distance map. According to the eigenvalues and eigenvectors of the Hessian matrix, the skeleton seed points are detected. Staring from the seed points, the initial centrelines are obtained using ridge traversal procedure. After that, we use minimum spanning tree to organize the geometrical structure of the skeleton points, and then we use graph trimming post-processing to compute the final centreline. Experimental results on different datasets demonstrate that our approach has high reliability, good robustness and requires less user interaction.

  20. Characterization of arbitrary fiber taper profiles with optical microscopy and image processing algorithms

    NASA Astrophysics Data System (ADS)

    Farias, Heric D.; Sebem, Renan; Paterno, Aleksander S.

    2014-08-01

    This work reports results from the development of a software to process the parameters involved in the characterization of fiber taper profiles, while using optical microscopy, a high-definition camera and a high- precision translation stage as the moveable base on which the taper is positioned. In addition to this procedure, image processing algorithms were customized to process the acquired images. With edge detection algorithms in the stitched image, one would be able to characterize the given taper radius curve that represents the taper profile when the camera has a sufficient resolution. As a consequence, the proposed fiber taper characterization procedure is a first step towards a high-resolution characterization of fiber taper diameters with arbitrary profiles, specially this case, in which tapers are fabricated with the stepwise technique that allows the production of non- biconical profiles. The parameters of the stitched images depends on the used microscope objective and the length of the characterized tapers. A non-biconical arbitrary taper is measured as an example for the illustration of the developed software and procedure.

  1. Asymmetric-detection time-stretch optical microscopy (ATOM) for ultrafast high-contrast cellular imaging in flow.

    PubMed

    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

    2014-01-13

    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.

  2. 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

  3. 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

  4. Analytic 3D imaging of mammalian nucleus at nanoscale using coherent x-rays and optical fluorescence microscopy.

    PubMed

    Song, Changyong; Takagi, Masatoshi; Park, Jaehyun; Xu, Rui; Gallagher-Jones, Marcus; Imamoto, Naoko; Ishikawa, Tetsuya

    2014-09-01

    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.

  5. 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

  6. 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

  7. Innovations of wide-field optical-sectioning fluorescence microscopy: toward high-speed volumetric bio-imaging with simplicity

    NASA Astrophysics Data System (ADS)

    Yu, Jiun-Yann

    Optical microscopy has become an indispensable tool for biological researches since its invention, mostly owing to its sub-cellular spatial resolutions, non-invasiveness, instrumental simplicity, and the intuitive observations it provides. Nonetheless, obtaining reliable, quantitative spatial information from conventional wide-field optical microscopy is not always intuitive as it appears to be. This is because in the acquired images of optical microscopy the information about out-of-focus regions is spatially blurred and mixed with in-focus information. In other words, conventional wide-field optical microscopy transforms the three-dimensional spatial information, or volumetric information about the objects into a two-dimensional form in each acquired image, and therefore distorts the spatial information about the object. Several fluorescence holography-based methods have demonstrated the ability to obtain three-dimensional information about the objects, but these methods generally rely on decomposing stereoscopic visualizations to extract volumetric information and are unable to resolve complex 3-dimensional structures such as a multi-layer sphere. The concept of optical-sectioning techniques, on the other hand, is to detect only two-dimensional information about an object at each acquisition. Specifically, each image obtained by optical-sectioning techniques contains mainly the information about an optically thin layer inside the object, as if only a thin histological section is being observed at a time. Using such a methodology, obtaining undistorted volumetric information about the object simply requires taking images of the object at sequential depths. Among existing methods of obtaining volumetric information, the practicability of optical sectioning has made it the most commonly used and most powerful one in biological science. However, when applied to imaging living biological systems, conventional single-point-scanning optical-sectioning techniques often

  8. 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.

  9. Cell segmentation in phase contrast microscopy images via semi-supervised classification over optics-related features.

    PubMed

    Su, Hang; Yin, Zhaozheng; Huh, Seungil; Kanade, Takeo

    2013-10-01

    Phase-contrast microscopy is one of the most common and convenient imaging modalities to observe long-term multi-cellular processes, which generates images by the interference of lights passing through transparent specimens and background medium with different retarded phases. Despite many years of study, computer-aided phase contrast microscopy analysis on cell behavior is challenged by image qualities and artifacts caused by phase contrast optics. Addressing the unsolved challenges, the authors propose (1) a phase contrast microscopy image restoration method that produces phase retardation features, which are intrinsic features of phase contrast microscopy, and (2) a semi-supervised learning based algorithm for cell segmentation, which is a fundamental task for various cell behavior analysis. Specifically, the image formation process of phase contrast microscopy images is first computationally modeled with a dictionary of diffraction patterns; as a result, each pixel of a phase contrast microscopy image is represented by a linear combination of the bases, which we call phase retardation features. Images are then partitioned into phase-homogeneous atoms by clustering neighboring pixels with similar phase retardation features. Consequently, cell segmentation is performed via a semi-supervised classification technique over the phase-homogeneous atoms. Experiments demonstrate that the proposed approach produces quality segmentation of individual cells and outperforms previous approaches.

  10. 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.

  11. 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

  12. Differentiating the extent of cartilage repair in rabbit ears using nonlinear optical microscopy.

    PubMed

    Zhu, X Q; Xu, Y H; Liao, C X; Liu, W G; Cheng, K K; Chen, J X

    2015-11-01

    Nonlinear optical microscopy (NLOM) was used as a noninvasive and label-free tool to detect and quantify the extent of the cartilage recovery. Two cartilage injury models were established in the outer ears of rabbits that created a different extent of cartilage recovery based on the presence or absence of the perichondrium. High-resolution NLOM images were used to measure cartilage repair, specifically through spectral analysis and image texture. In contrast to a wound lacking a perichondrium, wounds with intact perichondria demonstrated significantly larger TPEF signals from cells and matrix, coarser texture indicating the more deposition of type I collagen. Spectral analysis of cells and matrix can reveal the matrix properties and cell growth. In addition, texture analysis of NLOM images showed significant differences in the distribution of cells and matrix of repaired tissues with or without perichondrium. Specifically, the decay length of autocorrelation coefficient based on TPEF images is 11.2 ± 1.1 in Wound 2 (with perichondrium) and 7.5 ± 2.0 in Wound 1 (without perichondrium), indicating coarser image texture and faster growth of cells in repaired tissues with perichondrium (p < 0.05). Moreover, the decay length of autocorrelation coefficient based on collagen SHG images also showed significant difference between Wound 2 and 1 (16.2 ± 1.2 vs. 12.2 ± 2.1, p < 0.05), indicating coarser image texture and faster deposition of collagen in repaired tissues with perichondrium (Wound 2). These findings suggest that NLOM is an ideal tool for studying cartilage repair, with potential applications in clinical medicine. NLOM can capture macromolecular details and distinguish between different extents of cartilage repair without the need for labelling agents.

  13. Longitudinal label-free optical-resolution photoacoustic microscopy of tumor angiogenesis in vivo

    PubMed Central

    Lin, Riqiang; Chen, Jianhua; Wang, Huina; Yan, Meng; Zheng, Wei

    2015-01-01

    Background Optical-resolution photoacoustic microscopy (OR-PAM) is a high-resolution imaging technology capable of label-free imaging of the morphology and functions of the microvasculature in vivo. Previous studies of angiogenesis by OR-PAM were carried out primarily with transgenic mice and the mouse ear model. While important findings have been generated using this approach, the application of OR-PAM to the more widely used subcutaneous dorsal tumor models remains challenging, largely due to the respiratory and cardiac motion artifacts, as well as the protruding tumor contours. Methods and materials A noninvasive dorsal skin-fold (N-DSF) model, along with adaptive z-scanning and a corresponding experimental protocol, is developed. Mammary carcinoma cells (4T1) were administered subcutaneously to the backs of female BALB/c mice for tumor inoculation. The mice were anesthetized using a mixture of isofluorane and oxygen. Results In vivo OR-PAM of angiogenesis with subcutaneous dorsal tumor models in mice has been demonstrated. To test the performance of this method, we have monitored the growth of 4T1 mouse mammary carcinoma in BALB/c mice over a period of 9 days. The major features of tumor angiogenesis, including the change of vascular tortuosity, the dilation of vessel diameters, and the increase of blood supply, have been clearly captured with OR-PAM. Conclusions In combination with N-DSF model, OR-PAM has demonstrated outstanding capacity to provide label-free monitoring of angiogenesis in tumor. Thus, OR-PAM is of great potential to find broad biomedical applications in the pathophysiological studies of tumor and the treatments for anti-angiogenesis. PMID:25694950

  14. Ethanol alters gene expression and cell organization during optic vesicle evagination.

    PubMed

    Santos-Ledo, A; Cavodeassi, F; Carreño, H; Aijón, J; Arévalo, R

    2013-10-10

    Ethanol has been described as a teratogen in vertebrate development. During early stages of brain formation, ethanol affects the evagination of the optic vesicles, resulting in synophthalmia or cyclopia, phenotypes where the optic vesicles partially or totally fuse. The mechanisms by which ethanol affects the morphogenesis of the optic vesicles are however largely unknown. In this study we make use of in situ hybridization, electron microscopy and immunohistochemistry to show that ethanol has profound effects on cell organization and gene expression during the evagination of the optic vesicles. Exposure to ethanol during early eye development alters the expression patterns of some genes known to be important for eye morphogenesis, such as rx3/1 and six3a. Furthermore, exposure to ethanol interferes with the acquisition of neuroepithelial features by the eye field cells, which is clear at ultrastructual level. Indeed, ethanol disrupts the acquisition of fusiform cellular shapes within the eye field. In addition, tight junctions do not form and retinal progenitors do not properly polarize, as suggested by the mis-localization and down-regulation of zo1. We also show that the ethanol-induced cyclopic phenotype is significantly different to that observed in cyclopic mutants, suggesting a complex effect of ethanol on a variety of targets. Our results show that ethanol not only disrupts the expression pattern of genes involved in retinal morphogenesis, such as rx3 and rx1, but also disrupts the changes in cell polarity that normally occur during eye field splitting. Thus, ethylic teratology seems to be related not only to modifications in gene expression and cell death but also to alterations in cell morphology.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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)

  3. Oxidation study by Mössbauer and optic microscopy of steels from boiler tubes used in sugar industry

    NASA Astrophysics Data System (ADS)

    Fajardo, M.; Pérez Alcázar, G. A.; Aguilar, Y.

    1998-08-01

    Optic microscopy and Mössbauer spectroscopy were used to study the fail and the inner rusted surface of two boiler tubes used in the sugar industry, respectively. The studied tubes, of the type ASTM A 192, were found to have cracks. By optic microscopy it was observed that the failure begins in the inner surface with circumferential cracking. Also, inside and around the surface close to the cracks a rusted layer was detected. Powder from these layers was collected for Mössbauer spectroscopy analysis. By this method the presence of two or three types of Fe oxides such as wüstite, magnetite and hematite, was proved. These results permit to conclude that the failure mechanism was the thermal fatigue due to a hot work in an O2 -rich vapor atmosphere. The rusted products are stable at high temperatures.

  4. 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.

  5. The impact of the detection angle on the quantitative measurement of hemoglobin oxygen saturation in optical-resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Wu, Ning; Li, Changhui

    2016-10-01

    Optical-resolution photoacoustic microscopy (OR-PAM) plays an important role in the quantitative measurement of hemoglobin oxygen saturation (SO2) at a single vessel level. In this study, we reported that the relative angle between light illumination and ultrasonic detection could have a significant impact on the SO2 measurement. Both simulation and phantom studies were provided, and this result will help the system design and result interpretation of the functional OR-PAM.

  6. Optical-resolution photoacoustic microscopy of the metabolic rate of oxygen in a mouse renal tumor model

    NASA Astrophysics Data System (ADS)

    Yeh, Chenghung; Hu, Song; Liang, Jinyang; Li, Lei; Soetikno, Brian; Lu, Zhi Hong; Sohn, Rebecca E.; Maslov, Konstantin; Arbeit, Jeffrey M.; Wang, Lihong V.

    2015-03-01

    We propose using noninvasive longitudinal optical-resolution photoacoustic microscopy (L-ORPAM) to quantify blood flow flux, oxygen saturation (sO2), and thereby the metabolic rate of oxygen (MRO2), for a renal tumor model in the same mouse over weeks to months. Experiments showed that the sO2 difference between the artery and vein decreased greatly due to the arteriovenous shunting effect during tumor growth. Moreover, hypermetabolism was exhibited by an increase in MRO2.

  7. 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.

  8. Observer Performance in the Use of Digital and Optical Microscopy for the Interpretation of Tissue-Based Biomarkers

    PubMed Central

    Gavrielides, Marios A.; Conway, Catherine; O'Flaherty, Neil; Gallas, Brandon D.; Hewitt, Stephen M.

    2014-01-01

    Background. We conducted a validation study of digital pathology for the quantitative assessment of tissue-based biomarkers with immunohistochemistry. Objective. To examine observer agreement as a function of viewing modality (digital versus optical microscopy), whole slide versus tissue microarray (TMA) review, biomarker type (HER2 incorporating membranous staining and Ki-67 with nuclear staining), and data type (continuous and categorical). Methods. Eight pathologists reviewed 50 breast cancer whole slides (25 stained with HER2 and 25 with Ki-67) and 2 TMAs (1 stained with HER2, 1 with Ki-67, each containing 97 cores), using digital and optical microscopy. Results. Results showed relatively high overall interobserver and intermodality agreement, with different patterns specific to biomarker type. For HER2, there was better interobserver agreement for optical compared to digital microscopy for whole slides as well as better interobserver and intermodality agreement for TMAs. For Ki-67, those patterns were not observed. Conclusions. The differences in agreement patterns when examining different biomarkers and different scoring methods and reviewing whole slides compared to TMA stress the need for validation studies focused on specific pathology tasks to eliminate sources of variability that might dilute findings. The statistical uncertainty observed in our analyses calls for adequate sampling for each individual task rather than pooling cases. PMID:25763314

  9. 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.

  10. Adaptive optics in nonlinear microscopy implemented with open-loop control and EMCCD-based Shack-Hartmann wavefront sensor

    NASA Astrophysics Data System (ADS)

    Sun, Wei

    Nonlinear microscopy, with its unique advantages over conventional confocal fluorescence microscopy, has been widely adopted to study biological processes at the cellular level. However, like all other high-resolution optical imaging techniques, nonlinear microscopy suffers from focal degradation due to optical aberrations in the sample as a result of refractive index mismatch. Optical aberrations distort the wavefront of the excitation beam, causing the focal spot to be larger than the diffraction limit. Since the fluorescence efficiency scales nonlinearly with the profile of the focusing excitation beam, aberrations further degrade the image brightness in addition to resolution. In this dissertation I describe the design, characterization and experimentation of an adaptive optics (AO) nonlinear laser scanning microscope implemented with open-loop control and an EMCCD-based Shack-Hartmann wavefront sensor (EMCCD SHWFS) for aberration compensation. Adaptive optics (AO), originally designed for ground-based astronomical observatories to correct for the aberrations from atmospheric turbulence while imaging distant stars and planets, has benefited many biomedical imaging platforms. We integrated a microelectromechanical system (MEMS) deformable mirror (DM) into our nonlinear laser scanning microscope. With an accurate open-loop control mechanism, which predicts the control voltages and generates a prescribed surface shape on the MEMS DM, known aberrations in the system can be compensated for with this computationally simple and inherently fast method. The use of a nonlinear guide star imbedded within the sample can reflect the sample aberration. However, the low level of nonlinear fluorescence signal is usually detected by photomultiplier tubes (PMT) and is below the sensitivity of a conventional charge-coupled device (CCD) based Shack-Hartmann wavefront sensor. This dissertation also describes the design of an EMCCD SHWFS to measure the wavefront distortion from the

  11. Characterization of nodular and thermal defects in hafnia/silica multilayer coatings using optical, photothermal, and atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Stolz, Christopher J.; Yoshiyama, J. M.; Salleo, Alberto; Wu, Zhouling; Green, John; Krupka, Rene

    1998-04-01

    Multilayer coatings manufactured from metallic hafnium and silica sources by reactive electron beam deposition, are being developed for high fluence optics in a fusion lasers with a wavelength of 1053 nm and a 3 ns pulse length. Damage threshold studies have revealed a correlation between laser damage and nodular defects, but interestingly laser damage is also present in nodule-free regions. Photothermal studies of optical coatings reveal the existence of defects with strong optical absorption in nodule-free regions of the coating. A variety of microscopic techniques were employed to characterize the defects for a better understanding of the thermal properties of nodular defects and role of thermal defects in laser damage. Photothermal microscopy, utilizing the surface thermal lensing technique, was used to map the thermal characteristics of 3 mm X 3 mm areas of the coatings. High resolution subaperture scans, with a 1 micrometers step size and a 3 micrometers pump beam diameter, were conducted on the defects to characterize their photothermal properties. Optical and atomic force microscopy was used to visually identify defects and characterize their topography. The defects were then irradiated to determine the role of nodular and thermal defects in limiting the damage threshold of the multilayer.

  12. Characterization of nodular and thermal defects in hafnia/silica multilayer coatings using optical, photothermal, and atomic force microscopy

    SciTech Connect

    Stolz, C.J.; Yoshiyama, J.M.; Salleo, A.; Wu, Z.L.; Green, J.; Krupka, R.

    1997-12-24

    Multilayer coatings manufactured from metallic hafnium and silica sources by reactive electron beam deposition, are being developed for high fluence optics in a fusion laser with a wavelength of 1053 nm and a 3 ns pulse length. Damage threshold studies have revealed a correlation between laser damage and nodular defects, but interestingly laser damage is also present in nodule-free regions. Photothermal studies of optical coatings reveal the existence of defects with strong optical absorption in nodule-free regions of the coating. A variety of microscopic techniques were employed to characterize the effects for a better understanding of the thermal properties of nodular defects and role of thermal defects in laser damage. Photothermal microscopy, utilizing the surface thermal lensing technique, was used to map the thermal characteristics of 3 mm x 3 mm areas of the coatings. High resolution subaperture scans, with a 1 pm step size and a 3 um pump beam diameter, W= conducted on the defects to characterize their photothermal properties. Optical and atomic force microscopy was used to visually identify defects and characterize their topography. The defects were then irradiated to determine the role of nodular and thermal defects in limiting the damage threshold of the multilayer.

  13. Two-dimensional scanning focused refractive-index microscopy and applications to refractive-index profiling of optical fibers.

    PubMed

    Wang, Xiaowan; Ye, Qing; Sun, Tengqian; Wang, Jin; Deng, Zhichao; Mei, Jianchun; Zhou, Wenyuan; Zhang, Chunping; Tian, Jianguo

    2015-01-01

    The refractive-index profile (RIP) of optical fibers is of fundamental significance in determining critical fiber properties. Here, we present the application of a two-dimensional (2-D) scanning focused refractive-index microscopy (SFRIM) to accurately obtain the 2-D RIP of a graded-index optical fiber. Some modifications are made to SFRIM for better 2-D measurement. Quantitative RIP of the fiber is obtained with derivative total reflection method. The refractive-index accuracy is 0.002. The measured result is in good agreement with theoretical expectation. This method is straightforward, simple, repeatable, and free from signal distortion. This technique is suitable for symmetric and asymmetric optical fibers. The results indicate that this technique can be applied to obtain the RIPs of a wide range of materials and has broad application prospect in many fields.

  14. Determining the molecular origin of radiation damage/enhancement in electro-optic polymeric materials through polarized light microscopy

    NASA Astrophysics Data System (ADS)

    Perez-Moreno, Javier

    2014-09-01

    Previous studies on the radiation effects upon polymer and polymer-based photonic materials suggest that the radiation resistance of the material is heavily dependent on the choice of polymer-host and guest-chromophore. The best results to date have been achieved with electro optic polymeric materials based on CLD1 doped in APC, which has resulted in improved performance at the device level upon gamma-ray irradiation at moderate doses. Still, our understanding of the physical mechanisms behind the enhancement of the performance is unclear. In this paper, we discuss how polarized light microscopy could be used as a means to quantify the effect of the different physical parameters that influence the optical response of electro-optic polymeric thin film samples.

  15. A spatio-temporally compensated acousto-optic scanner for two-photon microscopy providing large field of view.

    PubMed

    Kremer, Y; Léger, J-F; Lapole, R; Honnorat, N; Candela, Y; Dieudonné, S; Bourdieu, L

    2008-07-01

    Acousto-optic deflectors (AOD) are promising ultrafast scanners for non-linear microscopy. Their use has been limited until now by their small scanning range and by the spatial and temporal dispersions of the laser beam going through the deflectors. We show that the use of AOD of large aperture (13mm) compared to standard deflectors allows accessing much larger field of view while minimizing spatio-temporal distortions. An acousto-optic modulator (AOM) placed at distance of the AOD is used to compensate spatial and temporal dispersions. Fine tuning of the AOM-AOD setup using a frequency-resolved optical gating (GRENOUILLE) allows elimination of pulse front tilt whereas spatial chirp is minimized thanks to the large aperture AOD.

  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. 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.

  18. 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.

  19. 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

  20. Revealing T-Tubules in Striated Muscle with New Optical Super-Resolution Microscopy Techniquess

    PubMed Central

    Clowsley, Alexander H.; Munro, Michelle; Hou, Yufeng; Crossman, David J.

    2014-01-01

    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

  1. Optical fiber nanoprobe preparation for near-field optical microscopy by chemical etching under surface tension and capillary action.

    PubMed

    Mondal, Samir K; Mitra, Anupam; Singh, Nahar; Sarkar, S N; Kapur, Pawan

    2009-10-26

    We propose a technique of chemical etching for fabrication of near perfect optical fiber nanoprobe (NNP). It uses photosensitive single mode optical fiber to etch in hydro fluoric (HF) acid solution. The difference in etching rate for cladding and photosensitive core in HF acid solution creates capillary ring along core-cladding boundary under a given condition. The capillary ring is filled with acid solution due to surface tension and capillary action. Finally it creates near perfect symmetric tip at the apex of the fiber as the height of the acid level in capillary ring decreases while width of the ring increases with continuous etching. Typical tip features are short taper length (approximately 4 microm), large cone angle (approximately 38 degrees ), and small probe tip dimension (<100 nm). A finite difference time domain (FDTD) analysis is also presented to compare near field optics of the NNP with conventional nanoprobe (CNP). The probe may be ideal for near field optical imaging and sensor applications.

  2. 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.

  3. Optical spectroscopy and microscopy of radiation-induced light-emitting point defects in lithium fluoride crystals and films

    NASA Astrophysics Data System (ADS)

    Montereali, R. M.; Bonfigli, F.; Menchini, F.; Vincenti, M. A.

    2012-08-01

    Broad-band light-emitting radiation-induced F2 and F3+ electronic point defects, which are stable and laser-active at room temperature in lithium fluoride crystals and films, are used in dosimeters, tuneable color-center lasers, broad-band miniaturized light sources and novel radiation imaging detectors. A brief review of their photoemission properties is presented, and their behavior at liquid nitrogen temperatures is discussed. Some experimental data from optical spectroscopy and fluorescence microscopy of these radiation-induced point defects in LiF crystals and thin films are used to obtain information about the coloration curves, the efficiency of point defect formation, the effects of photo-bleaching processes, etc. Control of the local formation, stabilization, and transformation of radiation-induced light-emitting defect centers is crucial for the development of optically active micro-components and nanostructures. Some of the advantages of low temperature measurements for novel confocal laser scanning fluorescence microscopy techniques, widely used for spatial mapping of these point defects through the optical reading of their visible photoluminescence, are highlighted.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. Aquagenic wrinkling of the palms: morphological changes in reflectance confocal microscopy and high-definition optical coherence tomography.

    PubMed

    Bielicky, Lea; Braun-Falco, Markus; Ruzicka, Thomas; Maier, Tanja

    2015-01-01

    Aquagenic wrinkling of the palms (AWP) is a rare condition, which is characterized by appearance of whitish papules and plaques, and an excessive wrinkling and swelling of the palmar skin after exposure to water. In most cases, young women are affected, and an association of AWP with cystic fibrosis (CF) has been surmised. We report on two cases of AWP, which were not related to CF, in whom we used two innovative imaging techniques, namely high-definition optical coherence tomography and reflectance confocal microscopy, to show in vivo skin changes occurring after exposure of the skin to tap water in comparison to the findings in a healthy control person.

  13. Electro-optical system for scanning microscopy of extreme ultraviolet masks with a high harmonic generation source.

    PubMed

    Naulleau, Patrick P; Anderson, Christopher N; Anderson, Erik H; Andreson, Nord; Chao, Weilun; Choi, Changhoon; Goldberg, Kenneth A; Gullikson, Eric M; Kim, Seong-Sue; Lee, Donggun; Miyakawa, Ryan; Park, Jongju; Rekawa, Seno; Salmassi, Farhad

    2014-08-25

    A self-contained electro-optical module for scanning extreme ultraviolet (EUV) reflection microscopy at 13.5 nm wavelength has been developed. The system has been designed to work with stand-alone commercially available EUV high harmonic generation (HHG) sources through the implementation of narrowband harmonic selecting multilayers and off-axis elliptical short focal length zoneplates. The module has been successfully integrated into an EUV mask scanning microscope achieving diffraction limited imaging performance (84 nm point spread function). PMID:25321224

  14. 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

  15. 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.

  16. 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)

  17. 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.

  18. 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.

  19. 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

  20. BigNeuron: Large-Scale 3D Neuron Reconstruction from Optical Microscopy Images.

    PubMed

    Peng, Hanchuan; Hawrylycz, Michael; Roskams, Jane; Hill, Sean; Spruston, Nelson; Meijering, Erik; Ascoli, Giorgio A

    2015-07-15

    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.

  1. Optical imaging of non-fluorescent nanodiamonds in live cells using transient absorption microscopy.

    PubMed

    Chen, Tao; Lu, Feng; Streets, Aaron M; Fei, Peng; Quan, Junmin; Huang, Yanyi

    2013-06-01

    We directly observe non-fluorescent nanodiamonds in living cells using transient absorption microscopy. This label-free technology provides a novel modality to study the dynamic behavior of nanodiamonds inside the cells with intrinsic three-dimensional imaging capability. We apply this method to capture the cellular uptake of nanodiamonds under various conditions, confirming the endocytosis mechanism.

  2. Extending resolution of scanning optical microscopy beyond the Abbe limit through the assistance of InSb thin layers.

    PubMed

    Ding, Chenliang; Wei, Jingsong; Li, Qisong; Liang, Xin; Wei, Tao

    2016-04-01

    The resolution of light imaging is required to extend beyond the Abbe limit to the subdiffraction, or even nanoscale. In this Letter, we propose to extend the resolution of scanning optical microscopy (SOM) beyond the Abbe limit as a kind of subdiffraction imaging technology through the assistance of InSb thin layers due to obvious nonlinear saturation absorption and reversible formation of an optical pinhole channel. The results show that the imaging resolution is greatly improved compared with the SOM itself. This work provides a way to improve the resolution of SOM without changing the SOM itself, but through the assistance of InSb thin layers. This is also a simple and practical way to extend the resolution of SOM beyond the Abbe limit.

  3. 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.

  4. 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

  5. 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

  6. Quantitative monitoring of the Chlamydia trachomatis developmental cycle using GFP-expressing bacteria, microscopy and flow cytometry.

    PubMed

    Vromman, François; Laverrière, Marc; Perrinet, Stéphanie; Dufour, Alexandre; Subtil, Agathe

    2014-01-01

    Chlamydiae are obligate intracellular bacteria. These pathogens develop inside host cells through a biphasic cycle alternating between two morphologically distinct forms, the infectious elementary body and the replicative reticulate body. Recently, C. trachomatis strains stably expressing fluorescent proteins were obtained. The fluorochromes are expressed during the intracellular growth of the microbe, allowing bacterial visualization by fluorescence microscopy. Whether they are also present in the infectious form, the elementary body, to a detectable level has not been studied. Here, we show that a C. trachomatis strain transformed with a plasmid expressing the green fluorescent protein (GFP) accumulates sufficient quantities of the probe in elementary bodies for detection by microscopy and flow cytometry. Adhesion of single bacteria was detected. The precise kinetics of bacterial entry were determined by microscopy using automated procedures. We show that during the intracellular replication phase, GFP is a convenient read-out for bacterial growth with several advantages over current methods. In particular, infection rates within a non-homogenous cell population are easily quantified. Finally, in spite of their small size, individual elementary bodies are detected by flow cytometers, allowing for direct enumeration of a bacterial preparation. In conclusion, GFP-expressing chlamydiae are suitable to monitor, in a quantitative manner, progression throughout the developmental cycle. This will facilitate the identification of the developmental steps targeted by anti-chlamydial drugs or host factors.

  7. Invited review article: combining scanning probe microscopy with optical spectroscopy for applications in biology and materials science.

    PubMed

    Lucas, Marcel; Riedo, Elisa

    2012-06-01

    This is a comprehensive review of the combination of scanning probe microscopy (SPM) with various optical spectroscopies, with a particular focus on Raman spectroscopy. Efforts to combine SPM with optical spectroscopy will be described, and the technical difficulties encountered will be examined. These efforts have so far focused mainly on the development of tip-enhanced Raman spectroscopy, a powerful technique to detect and image chemical signatures with single molecule sensitivity, which will be reviewed. Beyond tip-enhanced Raman spectroscopy and/or topography measurements, combinations of SPM with optical spectroscopy have a great potential in the characterization of structure and quantitative measurements of physical properties, such as mechanical, optical, or electrical properties, in delicate biological samples and nanomaterials. The different approaches to improve the spatial resolution, the chemical sensitivity, and the accuracy of physical properties measurements will be discussed. Applications of such combinations for the characterization of structure, defects, and physical properties in biology and materials science will be reviewed. Due to the versatility of SPM probes for the manipulation and characterization of small and/or delicate samples, this review will mainly focus on the apertureless techniques based on SPM probes.

  8. Out-of-focus background subtraction for fast structured illumination super-resolution microscopy of optically thick samples.

    PubMed

    Vermeulen, P; Zhan, H; Orieux, F; Olivo-Marin, J-C; Lenkei, Z; Loriette, V; Fragola, A

    2015-09-01

    We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three-dimensional (3D) samples that allows the use of two-dimensional (2D) data processing. Indeed, obtaining super-resolution images of thick samples is a difficult task if low spatial frequencies are present in the in-focus section of the sample, as these frequencies have to be distinguished from the out-of-focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high-resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out-of-focus content from the raw images. After this cleaning step, we can obtain super-resolution images of optical sections in thick samples using a two-beam harmonic illumination pattern and a limited number of raw images. This two-step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two-dimensional.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. Smart-phone based computational microscopy using multi-frame contact imaging on a fiber-optic array.

    PubMed

    Navruz, Isa; Coskun, Ahmet F; Wong, Justin; Mohammad, Saqib; Tseng, Derek; Nagi, Richie; Phillips, Stephen; Ozcan, Aydogan

    2013-10-21

    We demonstrate a cellphone based contact microscopy platform, termed Contact Scope, which can image highly dense or connected samples in transmission mode. Weighing approximately 76 grams, this portable and compact microscope is installed on the existing camera unit of a cellphone using an opto-mechanical add-on, where planar samples of interest are placed in contact with the top facet of a tapered fiber-optic array. This glass-based tapered fiber array has ~9 fold higher density of fiber optic cables on its top facet compared to the bottom one and is illuminated by an incoherent light source, e.g., a simple light-emitting-diode (LED). The transmitted light pattern through the object is then sampled by this array of fiber optic cables, delivering a transmission image of the sample onto the other side of the taper, with ~3× magnification in each direction. This magnified image of the object, located at the bottom facet of the fiber array, is then projected onto the CMOS image sensor of the cellphone using two lenses. While keeping the sample and the cellphone camera at a fixed position, the fiber-optic array is then manually rotated with discrete angular increments of e.g., 1-2 degrees. At each angular position of the fiber-optic array, contact images are captured using the cellphone camera, creating a sequence of transmission images for the same sample. These multi-frame images are digitally fused together based on a shift-and-add algorithm through a custom-developed Android application running on the smart-phone, providing the final microscopic image of the sample, visualized through the screen of the phone. This final computation step improves the resolution and also removes spatial artefacts that arise due to non-uniform sampling of the transmission intensity at the fiber optic array surface. We validated the performance of this cellphone based Contact Scope by imaging resolution test charts and blood smears.

  14. 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.

  15. Three dimensional time lapse imaging of live cell mitochondria with photothermal optical lock-in optical coherence microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Sison, Miguel; Chakrabortty, Sabyasachi; Extermann, Jerome; Nahas, Amir; Pache, Christophe; Weil, Tanja; Lasser, Theo

    2016-03-01

    The photothermal optical lock-in optical coherence microscope (poli-OCM) introduced molecular specificity to OCM imaging, which is conventionally, a label-free technique. Here we achieve three-dimensional live cell and mitochondria specific imaging using ~4nm protein-functionalized gold nanoparticles (AuNPs). These nanoparticles do not photobleach and we demonstrate they're suitability for long-term time lapse imaging. We compare the accuracy of labelling with these AuNPs using classical fluorescence confocal imaging with a standard mitochondria specific marker. Furthermore, time lapse poli-OCM imaging every 5 minutes over 1.5 hours period was achieved, revealing the ability for three-dimensional monitoring of mitochondria dynamics.

  16. 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

  17. Laser scanning confocal microscopy: history, applications, and related optical sectioning techniques.

    PubMed

    Paddock, Stephen W; Eliceiri, Kevin W

    2014-01-01

    Confocal microscopy is an established light microscopical technique for imaging fluorescently labeled specimens with significant three-dimensional structure. Applications of confocal microscopy in the biomedical sciences include the imaging of the spatial distribution of macromolecules in either fixed or living cells, the automated collection of 3D data, the imaging of multiple labeled specimens and the measurement of physiological events in living cells. The laser scanning confocal microscope continues to be chosen for most routine work although a number of instruments have been developed for more specific applications. Significant improvements have been made to all areas of the confocal approach, not only to the instruments themselves, but also to the protocols of specimen preparation, to the analysis, the display, the reproduction, sharing and management of confocal images using bioinformatics techniques. PMID:24052346

  18. Optical coherence microscopy for the in-depth study of biological structures: system based on a parallel detection scheme

    NASA Astrophysics Data System (ADS)

    Beaurepaire, Emmanuel; Gleyzes, Philippe; Lebec, Martial; Blanchot, Loic; Saint-Jalmes, Herve; Boccara, Albert C.

    1998-04-01

    We present a new microscopy system based on the OCT principle, that uses a multiplexed lock-in detection scheme to generate a 2D head-on image in parallel without lateral scanning. Our 'full-field optical coherence microscopy' comprises a Michelson interferometer built with a polarizing beam splitter, and uses a photoelastic birefringence modulator to modulate the optical path difference between the two orthogonal polarizations. A novel signal processing method is used to achieve a demodulation in parallel on every pixel of a 256 X 256 CCD camera. A 840 nm electroluminescent diode with 20 micrometers coherence length is used to illuminate the field of view through the microscope objective lens. In-depth exploration of the sample is realized by changing the plane of focus. The lateral resolution of the images is limited by the camera pixel size and is 2 micrometers . The axial sectioning ability is approximately 8 micrometers . Having validated our setup on model samples, we now evaluate its performance on biological structures. As an example, images of onion cells from 50-400 micrometers below the surface are obtained in 1 s with 100 dB sensitivity.

  19. High-efficiency localization of Na+-K+ ATPases on the cytoplasmic side by direct stochastic optical reconstruction microscopy

    NASA Astrophysics Data System (ADS)

    Wu, Jiazhen; Gao, Jing; Qi, Miao; Wang, Jianzhong; Cai, Mingjun; Liu, Shuheng; Hao, Xian; Jiang, Junguang; Wang, Hongda

    2013-11-01

    We describe a concise and effective strategy towards precisely mapping Na+-K+ ATPases on the cytoplasmic side of cell membranes by direct stochastic optical reconstruction microscopy (dSTORM). We found that most Na+-K+ ATPases are localized in different sizes of clusters on human red blood cell (hRBC) membranes, revealed by Ripley's K-function analysis. Further evidence that cholesterol depletion causes the dispersion of Na+-K+ ATPase clusters indicates that such clusters could be localized in cholesterol-enriched domains. Our results suggest that Na+-K+ ATPases might aggregate within the lipid rafts to fulfill their functions.We describe a concise and effective strategy towards precisely mapping Na+-K+ ATPases on the cytoplasmic side of cell membranes by direct stochastic optical reconstruction microscopy (dSTORM). We found that most Na+-K+ ATPases are localized in different sizes of clusters on human red blood cell (hRBC) membranes, revealed by Ripley's K-function analysis. Further evidence that cholesterol depletion causes the dispersion of Na+-K+ ATPase clusters indicates that such clusters could be localized in cholesterol-enriched domains. Our results suggest that Na+-K+ ATPases might aggregate within the lipid rafts to fulfill their functions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr03665k

  20. Defect-Assisted Hard-X-Ray Microscopy with Capillary Optics.

    PubMed

    Korecki, Paweł; Sowa, Katarzyna M; Jany, Benedykt R; Krok, Franciszek

    2016-06-10

    Polycapillary x-ray focusing devices are built from hundreds of thousands of bent microcapillaries that are stacked into hexagonal arrays. We show that intrinsic point defects of the optics (e.g., missing or larger capillaries) lead to the formation of multiple x-ray images of an object positioned in the focal plane. These images can be recorded in parallel, and can provide spatial resolution that is limited by the defect size and not by the focal spot size. In a proof-of-principle experiment, we demonstrate submicron resolution, which has not yet been achieved with polycapillary focusing optics. Tailored optics with a controlled distribution of "defects" could be used for multimodal nanoscale x-ray imaging with laboratory setups.

  1. Electro-Optical Imaging Microscopy of Dye-Doped Artificial Lipidic Membranes

    PubMed Central

    Hajj, Bassam; De Reguardati, Sophie; Hugonin, Loïc; Le Pioufle, Bruno; Osaki, Toshihisa; Suzuki, Hiroaki; Takeuchi, Shoji; Mojzisova, Halina; Chauvat, Dominique; Zyss, Joseph

    2009-01-01

    Artificial lipidic bilayers are widely used as a model for the lipid matrix in biological cell membranes. We use the Pockels electro-optical effect to investigate the properties of an artificial lipidic membrane doped with nonlinear molecules in the outer layer. We report here what is believed to be the first electro-optical Pockels signal and image from such a membrane. The electro-optical dephasing distribution within the membrane is imaged and the signal is shown to be linear as a function of the applied voltage. A theoretical analysis taking into account the statistical orientation distribution of the inserted dye molecules allows us to estimate the doped membrane nonlinearity. Ongoing extensions of this work to living cell membranes are discussed. PMID:19948120

  2. Analysis of the measured signals in apertureless near-field optical microscopy.

    PubMed

    Formanek, F; De Wilde, Y; Aigouy, L

    2005-05-01

    We present an analytical model able to explain the optical signal recorded during our experimental approach curves in the infrared at a wavelength lambda=10.6 microm, with a home-made apertureless near-field scanning optical microscope ANSOM. This model uses classical electrodynamics to calculate the scattering cross section of the oscillating tip, considered as a dipole, and its dielectric image in the sample as a function of the tip-sample separation from the near-field to the far-field regime. The dipoles are placed in a non-uniform electric field because of the standing wave arising from the interference between the incident and the specular laser beams. We also added a background field coming from a scatterer on the surface in order to account for zeroing of the optical signal for particular tip-sample separation and interference patterns.

  3. Fast spatial beam shaping by acousto-optic diffraction for 3D non-linear microscopy.

    PubMed

    Akemann, Walther; Léger, Jean-François; Ventalon, Cathie; Mathieu, Benjamin; Dieudonné, Stéphane; Bourdieu, Laurent

    2015-11-01

    Acousto-optic deflection (AOD) devices offer unprecedented fast control of the entire spatial structure of light beams, most notably their phase. AOD light modulation of ultra-short laser pulses, however, is not straightforward to implement because of intrinsic chromatic dispersion and non-stationarity of acousto-optic diffraction. While schemes exist to compensate chromatic dispersion, non-stationarity remains an obstacle. In this work we demonstrate an efficient AOD light modulator for stable phase modulation using time-locked generation of frequency-modulated acoustic waves at the full repetition rate of a high power laser pulse amplifier of 80 kHz. We establish the non-local relationship between the optical phase and the generating acoustic frequency function and verify the system for temporal stability, phase accuracy and generation of non-linear two-dimensional phase functions. PMID:26561090

  4. Fast spatial beam shaping by acousto-optic diffraction for 3D non-linear microscopy.

    PubMed

    Akemann, Walther; Léger, Jean-François; Ventalon, Cathie; Mathieu, Benjamin; Dieudonné, Stéphane; Bourdieu, Laurent

    2015-11-01

    Acousto-optic deflection (AOD) devices offer unprecedented fast control of the entire spatial structure of light beams, most notably their phase. AOD light modulation of ultra-short laser pulses, however, is not straightforward to implement because of intrinsic chromatic dispersion and non-stationarity of acousto-optic diffraction. While schemes exist to compensate chromatic dispersion, non-stationarity remains an obstacle. In this work we demonstrate an efficient AOD light modulator for stable phase modulation using time-locked generation of frequency-modulated acoustic waves at the full repetition rate of a high power laser pulse amplifier of 80 kHz. We establish the non-local relationship between the optical phase and the generating acoustic frequency function and verify the system for temporal stability, phase accuracy and generation of non-linear two-dimensional phase functions.

  5. Virtual-'Light-Sheet' Single-Molecule Localisation Microscopy Enables Quantitative Optical Sectioning for Super-Resolution Imaging

    PubMed Central

    Palayret, Matthieu; Armes, Helen; Basu, Srinjan; Watson, Adam T.; Herbert, Alex; Lando, David; Etheridge, Thomas J.; Endesfelder, Ulrike; Heilemann, Mike; Laue, Ernest; Carr, Antony M.; Klenerman, David; Lee, Steven F.

    2015-01-01

    Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated. PMID:25884495

  6. Remote optical sensing on the nanometer scale with a bowtie aperture nano-antenna on a fiber tip of scanning near-field optical microscopy

    SciTech Connect

    Atie, Elie M.; Xie, Zhihua; El Eter, Ali; Salut, Roland; Baida, Fadi I.; Grosjean, Thierry; Nedeljkovic, Dusan; Tannous, Tony

    2015-04-13

    Plasmonic nano-antennas have proven the outstanding ability of sensing chemical and physical processes down to the nanometer scale. Sensing is usually achieved within the highly confined optical fields generated resonantly by the nano-antennas, i.e., in contact to the nanostructures. In this paper, we demonstrate the sensing capability of nano-antennas to their larger scale environment, well beyond their plasmonic confinement volume, leading to the concept of “remote” (non contact) sensing on the nanometer scale. On the basis of a bowtie-aperture nano-antenna (BNA) integrated at the apex of a SNOM (Scanning Near-field Optical Microscopy) fiber tip, we introduce an ultra-compact, moveable, and background-free optical nanosensor for the remote sensing of a silicon surface (up to distance of 300 nm). Sensitivity of the BNA to its large scale environment is high enough to expect the monitoring and control of the spacing between the nano-antenna and a silicon surface with sub-nanometer accuracy. This work paves the way towards an alternative class of nanopositioning techniques, based on the monitoring of diffraction-free plasmon resonance, that are alternative to nanomechanical and diffraction-limited optical interference-based devices.

  7. Blind frequency-resolved optical-gating pulse characterization for quantitative differential multiphoton microscopy.

    PubMed

    Field, Jeffrey J; Durfee, Charles G; Squier, Jeff A

    2010-10-15

    We use a unique multifocal multiphoton microscope to directly characterize the pulse in the focal plane of a high-NA objective using second-harmonic generation frequency-resolved optical gating (FROG). Because of the nature of the optical setup, femtosecond laser pulses of orthogonal polarization states are generated in the focal plane, each acquiring a different spectral dispersion. By applying an additional constraint on the phase extraction algorithm, we simultaneously extract both the gate and probe pulses from a single spectrogram with a FROG error of 0.016. PMID:20967069

  8. Changes in ocular aquaporin expression following optic nerve crush

    PubMed Central

    Oku, Hidehiro; Fukuhara, Masayuki; Kurimoto, Takuji; Ikeda, Tsunehiko; Patil, Rajkumar V.; Sharif, Najam A.; Yorio, Thomas

    2010-01-01

    Purpose Changes in the expression of water channels (aquaporins; AQP) have been reported in several diseases. However, such changes and mechanisms remain to be evaluated for retinal injury after optic nerve crush (ONC). This study was designed to analyze changes in the expression of AQP4 (water selective channel) and AQP9 (water and lactate channel) following ONC in the rat. Methods Rat retinal ganglion cells (RGCs) were retrogradely labeled by applying FluoroGold onto the left superior colliculus 1 week before ONC. Retinal injuries were induced by ONC unilaterally. Real-time PCR was used to measure changes in AQP4, AQP9, thy-1, Kir4.1 (K+ channel), and β-actin messages. Changes in AQP4, AQP9, Kir4.1, B cell lymphoma-x (bcl-xl), and glial fibrillary acidic protein (GFAP) expression were measured in total retinal extracts using western blotting. Results The number of RGCs labeled retrogradely from the superior colliculus was 2,090±85 cells/mm2 in rats without any treatment, which decreased to 1,091±78 (47% loss) and 497±87 cells/mm2 (76% loss) on days 7 and 14, respectively. AQP4, Kir4.1, and thy-1 protein levels decreased at days 2, 7, and 14, which paralleled a similar reduction in mRNA levels, with the exception of Kir4.1 mRNA at day 2 showing an apparent upregulation. In contrast, AQP9 mRNA and protein levels showed opposite changes to those observed for the latter targets. Whereas AQP9 mRNA increased at days 2 and 14, protein levels decreased at both time points. AQP9 mRNA decreased at day 7, while protein levels increased. GFAP (a marker of astrogliosis) remained upregulated at days 2, 7, and 14, while bcl-xl (anti-apoptotic) decreased. Conclusions The reduced expression of AQP4 and Kir4.1 suggests dysfunctional ion coupling in retina following ONC and likely impaired retinal function. The sustained increase in GFAP indicates astrogliosis, while the decreased bcl-xl protein level suggests a commitment to cellular death, as clearly shown by the reduction in

  9. In vivo monitoring of seeds and plant-tissue water absorption using optical coherence tomography and optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Sapozhnikova, Veronika V.; Kutis, Irina S.; Kutis, Sergey D.; Kuranov, Roman V.; Gelikonov, Grigory V.; Shabanov, Dmitry V.; Kamensky, Vladislav A.

    2004-07-01

    First experimental results on OCT imaging of internal structure of plant tissues and in situ OCT monitoring of plant tissue regeneration at different water supply are reported. Experiments for evaluating OCT capabilities were performed on Tradescantia. The investigation of seeds swelling was performed on wheat seeds (Triticum L.), barley seeds (Hordeum L.), long-fibred flax seeds (Linum usitatissimum L.) and cucumber seeds (Cucumis sativus L.). These OCT images correlate with standard microscopy data from the same tissue regions. Seeds were exposed to a low-intensity physical factor-the pulsed gradient magnetic field (GMF) with pulse duration 0.1 s and maximum amplitude 5 mT (4 successive pulses during 0.4 s). OCT and OCM enable effective monitoring of fast reactions in plants and seeds at different water supply.

  10. Single-shot observation of optical rogue waves in integrable turbulence using time microscopy

    PubMed Central

    Suret, Pierre; Koussaifi, Rebecca El; Tikan, Alexey; Evain, Clément; Randoux, Stéphane; Szwaj, Christophe; Bielawski, Serge

    2016-01-01

    Optical fibres are favourable tabletop laboratories to investigate both coherent and incoherent nonlinear waves. In particular, exact solutions of the one-dimensional nonlinear Schrödinger equation such as fundamental solitons or solitons on finite background can be generated by launching periodic, specifically designed coherent waves in optical fibres. It is an open fundamental question to know whether these coherent structures can emerge from the nonlinear propagation of random waves. However the typical sub-picosecond timescale prevented—up to now—time-resolved observations of the awaited dynamics. Here, we report temporal ‘snapshots' of random light using a specially designed ‘time-microscope'. Ultrafast structures having peak powers much larger than the average optical power are generated from the propagation of partially coherent waves in optical fibre and are recorded with 250 femtoseconds resolution. Our experiment demonstrates the central role played by ‘breather-like' structures such as the Peregrine soliton in the emergence of heavy-tailed statistics in integrable turbulence. PMID:27713416

  11. Single-shot observation of optical rogue waves in integrable turbulence using time microscopy

    NASA Astrophysics Data System (ADS)

    Suret, Pierre; Koussaifi, Rebecca El; Tikan, Alexey; Evain, Clément; Randoux, Stéphane; Szwaj, Christophe; Bielawski, Serge

    2016-10-01

    Optical fibres are favourable tabletop laboratories to investigate both coherent and incoherent nonlinear waves. In particular, exact solutions of the one-dimensional nonlinear Schrödinger equation such as fundamental solitons or solitons on finite background can be generated by launching periodic, specifically designed coherent waves in optical fibres. It is an open fundamental question to know whether these coherent structures can emerge from the nonlinear propagation of random waves. However the typical sub-picosecond timescale prevented--up to now--time-resolved observations of the awaited dynamics. Here, we report temporal `snapshots' of random light using a specially designed `time-microscope'. Ultrafast structures having peak powers much larger than the average optical power are generated from the propagation of partially coherent waves in optical fibre and are recorded with 250 femtoseconds resolution. Our experiment demonstrates the central role played by `breather-like' structures such as the Peregrine soliton in the emergence of heavy-tailed statistics in integrable turbulence.

  12. Acousto-optic multiphoton laser scanning microscopy and multiphoton photon counting spectroscopy: Applications and implications for optical neurobiology

    NASA Astrophysics Data System (ADS)

    Iyer, Vijay

    Multiphoton excitation of molecular probes has become an important tool in experimental neurobiology owing to the intrinsic optical sectioning and low light scattering it affords. Using molecular functional indicators, multiphoton excitation allows physiological signals within single neurons to be observed from within living brain tissue. Ideally, it would be possible to record from multiple sites located throughout the elaborately branching dendritic arbors, in order to study the correlations of structure and function both within and across experiments. However, existing multiphoton microscope systems based on scanning mirrors do not allow optical recordings to be obtained from more than a handful of sites simultaneously at the high rates required to capture the fast physiological signals of interest (>100Hz for Ca2+ signals, >1kHz for membrane potential transients). In order to overcome this limitation, two-dimensional acousto-optic deflection was employed, to allow an ultrafast laser beam suited for multiphoton excitation to be rapidly repositioned with low latency (˜15mus). This supports a random-access scanning mode in which the beam can repeatedly visit a succession of user-selected sites of interest within the microscope's field-of-view at high rates, with minimal sacrifice of pixel dwell time. This technique of acousto-optic multiphoton laser scanning microscope (AO-MPLSM) was demonstrated to allow the spatial profile of signals arising in response to physiological stimulation to be rapidly mapped. Means to compensate or avoid problems of dispersion which have hampered AO-MPLSM in the past are presented, with the latter being implemented. Separately, the combination of photon counting detection with multiphoton excitation, termed generally multiphoton photon counting spectroscopy (MP-PCS), was also considered, with particular emphasis on the technique of fluorescence correlation spectroscopy (FCS). MP-PCS was shown to allow information about molecular

  13. Optical Stimulation of Zebrafish Hair Cells Expressing Channelrhodopsin-2

    PubMed Central

    Kreines, Fabiana; Trapani, Josef G.

    2014-01-01

    Vertebrate hair cells are responsible for the high fidelity encoding of mechanical stimuli into trains of action potentials (spikes) in afferent neurons. Here, we generated a transgenic zebrafish line expressing Channelrhodopsin-2 (ChR2) under the control of the hair-cell specific myo6b promoter, in order to examine the role of the mechanoelectrical transduction (MET) channel in sensory encoding in afferent neurons. We performed in vivo recordings from afferent neurons of the zebrafish lateral line while activating hair cells with either mechanical stimuli from a waterjet or optical stimuli from flashes of ∼470-nm light. Comparison of the patterns of encoded spikes during 100-ms stimuli revealed no difference in mean first spike latency between the two modes of activation. However, there was a significant increase in the variability of first spike latency during optical stimulation as well as an increase in the mean number of spikes per stimulus. Next, we compared encoding of spikes during hair-cell stimulation at 10, 20, and 40-Hz. Consistent with the increased variability of first spike latency, we saw a significant decrease in the vector strength of phase-locked spiking during optical stimulation. These in vivo results support a physiological role for the MET channel in the high fidelity of first spike latency seen during encoding of mechanical sensory stimuli. Finally, we examined whether remote activation of hair cells via ChR2 activation was sufficient to elicit escape responses in free-swimming larvae. In transgenic larvae, 100-ms flashes of ∼470-nm light resulted in escape responses that occurred concomitantly with field recordings indicating Mauthner cell activity. Altogether, the myo6b:ChR2 transgenic line provides a platform to investigate hair-cell function and sensory encoding, hair-cell sensory input to the Mauthner cell, and the ability to remotely evoke behavior in free-swimming zebrafish. PMID:24791934

  14. Raldh2 expression in optic vesicle generates a retinoic acid signal needed for invagination of retina during optic cup formation.

    PubMed

    Mic, Felix A; Molotkov, Andrei; Molotkova, Natalia; Duester, Gregg

    2004-10-01

    Three retinaldehyde dehydrogenase genes (Raldh1, Raldh2, and Raldh3) expressed in unique spatiotemporal patterns may control synthesis of retinoic acid (RA) needed for retina development. However, previous studies indicate that retina formation still proceeds normally in Raldh1-/- mouse embryos lacking RA synthesis in the dorsal neural retina at the optic cup stage. Here, we demonstrate that Raldh2-/- embryos lacking RA synthesis in the optic vesicle exhibit a failure in retina invagination needed to develop an optic cup. This was also observed in Raldh1-/-:Raldh2-/- double mutants, which develop similarly. Both mutants retain RA activity in the lens placode associated with Raldh3 expression, but this RA activity is insufficient to induce optic cup formation. Maternal RA administration at the optic vesicle stage rescues optic cup formation in Raldh2-/- and Raldh1-/-:Raldh2-/- embryos, demonstrating that Raldh1 is not required during rescue of optic cup development. The optic cup of rescued Raldh1-/-:Raldh2-/- embryos exhibits normal RA activity and this is associated with Raldh3 expression in the retina and lens. Thus, RA signaling initiates in the optic vesicle in response to Raldh2 but can be maintained during optic cup formation by a gene other than Raldh1, most likely Raldh3. Loss of optic vesicle RA signaling does not effect expression of early determinants of retina at the optic vesicle stage (Pax6, Six3, Rx, Mitf). Our findings suggest that RA functions as one of the signals needed for invagination of the retina to generate an optic cup. PMID:15366004

  15. Optical microscopy of Arabidopsis seedlings fixed in non-fresh FAA using Kennedy Fixation Tubes.

    PubMed

    Honma, Yoshihiro; Nakabayashi, Izumi; Tamaoki, Daisuke; Kasahara, Haruo; Ishioka, Noriaki; Shimazu, Toru; Kasahara, Hirokazu; Yamada, Mitsuhiro; Karahara, Ichirou; Kamisaka, Seiichiro

    2003-12-01

    Samples will be kept in non-fresh fixative for longer period than usual in Kennedy Space Center Fixation Tubes (KFT) when they will be fixed for microscopy in the International Space Station (ISS). It is necessary to examine characteristics of samples prepared under such conditions and to improve preservation of the samples. In this study, morphology of Arabidopsis tissues prepared under such conditions was examined under a light microscope. Deformation of cell shape was observed in tissues with well-developed intercellular spaces when they were fixed in non-fresh fixative for longer period in KFTs.

  16. Utilization of optical polarization microscopy in the study of sorption characteristics of wound dressing host materials.

    PubMed

    Devetak, Miha; Peršin, Zdenka; Stana-Kleinschek, Karin; Maver, Uroš

    2014-04-01

    Polarization microscopy was used for evaluation of kinetics of diclofenac sorption in three different wound dressing materials. The sorption kinetics can be evaluated by radii change and intensity of the light traveling through the fiber. The most frequently used host materials for drugs in wound dressings are alginate, polyesters such as polyethylene terephthalate, and viscose. We studied sorption of diclofenac as an example drug. Effective, but rather simple in vitro simulation of diclofenac sorption gives insight into the applicability of the mentioned materials for development of wound healing materials.

  17. Feasibility study on 3-D shape analysis of high-aspect-ratio features using through-focus scanning optical microscopy

    PubMed Central

    Attota, Ravi Kiran; Weck, Peter; Kramar, John A.; Bunday, Benjamin; Vartanian, Victor

    2016-01-01

    In-line metrologies currently used in the semiconductor industry are being challenged by the aggressive pace of device scaling and the adoption of novel device architectures. Metrology and process control of three-dimensional (3-D) high-aspect-ratio (HAR) features are becoming increasingly important and also challenging. In this paper we present a feasibility study of through-focus scanning optical microscopy (TSOM) for 3-D shape analysis of HAR features. TSOM makes use of 3-D optical data collected using a conventional optical microscope for 3-D shape analysis. Simulation results of trenches and holes down to the 11 nm node are presented. The ability of TSOM to analyze an array of HAR features or a single isolated HAR feature is also presented. This allows for the use of targets with area over 100 times smaller than that of conventional gratings, saving valuable real estate on the wafers. Indications are that the sensitivity of TSOM may match or exceed the International Technology Roadmap for Semiconductors (ITRS) measurement requirements for the next several years. Both simulations and preliminary experimental results are presented. The simplicity, lowcost, high throughput, and nanometer scale 3-D shape sensitivity of TSOM make it an attractive inspection and process monitoring solution for nanomanufacturing. PMID:27464112

  18. Feasibility study on 3-D shape analysis of high-aspect-ratio features using through-focus scanning optical microscopy.

    PubMed

    Attota, Ravi Kiran; Weck, Peter; Kramar, John A; Bunday, Benjamin; Vartanian, Victor

    2016-07-25

    In-line metrologies currently used in the semiconductor industry are being challenged by the aggressive pace of device scaling and the adoption of novel device architectures. Metrology and process control of three-dimensional (3-D) high-aspect-ratio (HAR) features are becoming increasingly important and also challenging. In this paper we present a feasibility study of through-focus scanning optical microscopy (TSOM) for 3-D shape analysis of HAR features. TSOM makes use of 3-D optical data collected using a conventional optical microscope for 3-D shape analysis. Simulation results of trenches and holes down to the 11 nm node are presented. The ability of TSOM to analyze an array of HAR features or a single isolated HAR feature is also presented. This allows for the use of targets with area over 100 times smaller than that of conventional gratings, saving valuable real estate on the wafers. Indications are that the sensitivity of TSOM may match or exceed the International Technology Roadmap for Semiconductors (ITRS) measurement requirements for the next several years. Both simulations and preliminary experimental results are presented. The simplicity, lowcost, high throughput, and nanometer scale 3-D shape sensitivity of TSOM make it an attractive inspection and process monitoring solution for nanomanufacturing. PMID:27464112

  19. Fabrication and Atomic Force Microscopy Characterization of Molecular Composites of Fullerenes in Aerogel Matrix for Optical Limiting

    NASA Technical Reports Server (NTRS)

    Lu, W. J .; Sunkara, H. B.; Shi, D.; Morgan, S. H.; Penn, B.; Frazier, D.; Collins, W. E.

    1998-01-01

    An optical limiter is a device which exhibits a decrease in the transmittance in a material with an increase in intensity of light. Sol-gel techniques offer many advantages in the fabrication of materials. These materials possess many desirable properties for nonlinear optical (NLO) device applications which include transparency, high thermal and chemical stabilities, very low refractive index and dielectric constants. C60 shows a higher excited state absorption cross section than the ground state absorption cross section over the complete visible spectrum, and the spectrum of the excited state absorption of C60 has the same general shape as the ground state absorption. This fact suggests that fullerenes are ideal optical limiting materials. Aerogels are fabricated by sol-gel processing. One of the key issues is the dispersion of fullerenes into small and uniform pores of silica aerogel host matrices. The aerogel network was characterized by Raman spectroscopy. Atomic force microscopy is a technique with many advantages to characterize the aerogel materials. The morphology of the cleaved surface for a C60/aerogel sample shows that there are long paralleled shaped stripes with 20-30 nm in width and about 500 nm in length on the cleaved surface. The cleaved surface also was etched by 5% HF solution for one minutes, and it became smoother after HF etching. The main feature in on the surface is the spherical particles with the size of few nanometers, and no aggregated fullerenes appear. The fullerenes are well dispersed in the aerogel matrices.

  20. Compressive sensing based high-speed time-stretch optical microscopy for two-dimensional image acquisition.

    PubMed

    Guo, Qiang; Chen, Hongwei; Weng, Zhiliang; Chen, Minghua; Yang, Sigang; Xie, Shizhong

    2015-11-16

    In this paper, compressive sensing based high-speed time-stretch optical microscopy for two-dimensional (2D) image acquisition is proposed and experimentally demonstrated for the first time. A section of dispersion compensating fiber (DCF) is used to perform wavelength-to-time conversion and then ultrafast spectral shaping of broadband optical pulses can be achieved via high-speed intensity modulation. A 2D spatial disperser comprising a pair of orthogonally oriented dispersers is employed to produce spatially structured illumination for 2D image acquisition and a section of single mode fiber (SMF) is utilized for pulse compression in the optical domain. In our scheme, a 1.2-GHz photodetector and a 50-MHz analog-to-digital converter (ADC) are used to acquire the energy of the compressed pulses. Image reconstructions are demonstrated at a frame rate of 500 kHz and a sixteen-fold image compression is achieved in our proof-of-concept demonstration.