Optics in the United kingdom.

PubMed

Ditchburn, R W

1969-10-01

Optics is interpreted to include x-ray optics, electronic optics, and short wave radiooptics as well as the more conventional visible, uv, and ir optics. Recent work in Britain on x-ray optics (applied to molecular biology), on scanning electron microscopy, and in radioastronomy (discovery of pulsars) is mentioned. In the optics of the visible and ir there is an increasing interest in over-all systems design. .The formation of large industrial units capable of carrying through major design program, requiring advanced mechanical and electronic design associated with new lens systems, is welcomed.

Optical toolkits for in vivo deep tissue laser scanning microscopy: a primer

NASA Astrophysics Data System (ADS)

Lee, Woei Ming; McMenamin, Thomas; Li, Yongxiao

2018-06-01

Life at the microscale is animated and multifaceted. The impact of dynamic in vivo microscopy in small animals has opened up opportunities to peer into a multitude of biological processes at the cellular scale in their native microenvironments. Laser scanning microscopy (LSM) coupled with targeted fluorescent proteins has become an indispensable tool to enable dynamic imaging in vivo at high temporal and spatial resolutions. In the last few decades, the technique has been translated from imaging cells in thin samples to mapping cells in the thick biological tissue of living organisms. Here, we sought to provide a concise overview of the design considerations of a LSM that enables cellular and subcellular imaging in deep tissue. Individual components under review include: long working distance microscope objectives, laser scanning technologies, adaptive optics devices, beam shaping technologies and photon detectors, with an emphasis on more recent advances. The review will conclude with the latest innovations in automated optical microscopy, which would impact tracking and quantification of heterogeneous populations of cells in vivo.

Correlating microscopy techniques and ToF-SIMS analysis of fully grown mammalian oocytes.

PubMed

Gulin, Alexander; Nadtochenko, Victor; Astafiev, Artyom; Pogorelova, Valentina; Rtimi, Sami; Pogorelov, Alexander

2016-06-20

The 2D-molecular thin film analysis protocol for fully grown mice oocytes is described using an innovative approach. Time-of-flight secondary ion mass spectrometry (ToF-SIMS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and optical microscopy imaging were applied to the same mice oocyte section on the same sample holder. A freeze-dried mice oocyte was infiltrated into embedding media, e.g. Epon, and then was cut with a microtome and 2 μm thick sections were transferred onto an ITO coated conductive glass. Mammalian oocytes can contain "nucleolus-like body" (NLB) units and ToF-SIMS analysis was used to investigate the NLB composition. The ion-spatial distribution in the cell components was identified and compared with the images acquired by SEM, AFM and optical microscopy. This study presents a significant advancement in cell embryology, cell physiology and cancer-cell biochemistry.

Measurement of time-varying displacement fields in cell culture for traction force optical coherence microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Mulligan, Jeffrey A.; Adie, Steven G.

2017-02-01

Mechanobiology is an emerging field which seeks to link mechanical forces and properties to the behaviors of cells and tissues in cancer, stem cell growth, and other processes. Traction force microscopy (TFM) is an imaging technique that enables the study of traction forces exerted by cells on their environment to migrate as well as sense and manipulate their surroundings. To date, TFM research has been performed using incoherent imaging modalities and, until recently, has been largely confined to the study of cell-induced tractions within two-dimensions using highly artificial and controlled environments. As the field of mechanobiology advances, and demand grows for research in physiologically relevant 3D culture and in vivo models, TFM will require imaging modalities that support such settings. Optical coherence microscopy (OCM) is an interferometric imaging modality which enables 3D cellular resolution imaging in highly scattering environments. Moreover, optical coherence elastography (OCE) enables the measurement of tissue mechanical properties. OCE relies on the principle of measuring material deformations in response to artificially applied stress. By extension, similar techniques can enable the measurement of cell-induced deformations, imaged with OCM. We propose traction force optical coherence microscopy (TF-OCM) as a natural extension and partner to existing OCM and OCE methods. We report the first use of OCM data and digital image correlation to track temporally varying displacement fields exhibited within a 3D culture setting. These results mark the first steps toward the realization of TF-OCM in 2D and 3D settings, bolstering OCM as a platform for advancing research in mechanobiology.

The light-sheet microscopy revolution

NASA Astrophysics Data System (ADS)

Girkin, J. M.; Carvalho, M. T.

2018-05-01

This paper reviews the rapid advances that have been made in one form of optical biological imaging in the last decade, namely that of light sheet microscopy. Although the concept was originally presented over one hundred years ago, at the time it was a methodology that lacked the technology to really make it a viable tool for practical everyday imaging in the biologist’s laboratory. However, since its re-discovery, it has started to transform in vivo and increasingly intact organ imaging in a number of areas of biology. This review looks back at the beginning of the method and then the crucial role that modern optical technology, frequently developed for other fields, has played in advancing the instrumentation. This paper will also look at the OpenSPIM route that was developed whereby, through the purchase of a few optical components, researchers have been able to develop their own bespoke instruments and we consider if this may be a route forward for the rapid development of other technological breakthroughs.

Advanced Analytical Methodologies Based on Raman Spectroscopy to Detect Prebiotic and Biotic Molecules: Applicability in the Study of the Martian Nakhlite NWA 6148 Meteorite

NASA Astrophysics Data System (ADS)

Madariaga, J. M.; Torre-Fdez, I.; Ruiz-Galende, P.; Aramendia, J.; Gomez-Nubla, L.; Fdez-Ortiz de Vallejuelo, S.; Maguregui, M.; Castro, K.; Arana, G.

2018-04-01

Advanced methodologies based on Raman spectroscopy are proposed to detect prebiotic and biotic molecules in returned samples from Mars: (a) optical microscopy with confocal micro-Raman, (b) the SCA instrument, (c) Raman Imaging. Examples for NWA 6148.

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.

Nanoscale live cell optical imaging of the dynamics of intracellular microvesicles in neural cells.

PubMed

Lee, Sohee; Heo, Chaejeong; Suh, Minah; Lee, Young Hee

2013-11-01

Recent advances in biotechnology and imaging technology have provided great opportunities to investigate cellular dynamics. Conventional imaging methods such as transmission electron microscopy, scanning electron microscopy, and atomic force microscopy are powerful techniques for cellular imaging, even at the nanoscale level. However, these techniques have limitations applications in live cell imaging because of the experimental preparation required, namely cell fixation, and the innately small field of view. In this study, we developed a nanoscale optical imaging (NOI) system that combines a conventional optical microscope with a high resolution dark-field condenser (Cytoviva, Inc.) and halogen illuminator. The NOI system's maximum resolution for live cell imaging is around 100 nm. We utilized NOI to investigate the dynamics of intracellular microvesicles of neural cells without immunocytological analysis. In particular, we studied direct, active random, and moderate random dynamic motions of intracellular microvesicles and visualized lysosomal vesicle changes after treatment of cells with a lysosomal inhibitor (NH4Cl). Our results indicate that the NOI system is a feasible, high-resolution optical imaging system for live small organelles that does not require complicated optics or immunocytological staining processes.

Planar Diffractive Lenses: Fundamentals, Functionalities, and Applications.

PubMed

Huang, Kun; Qin, Fei; Liu, Hong; Ye, Huapeng; Qiu, Cheng-Wei; Hong, Minghui; Luk'yanchuk, Boris; Teng, Jinghua

2018-06-01

Traditional objective lenses in modern microscopy, based on the refraction of light, are restricted by the Rayleigh diffraction limit. The existing methods to overcome this limit can be categorized into near-field (e.g., scanning near-field optical microscopy, superlens, microsphere lens) and far-field (e.g., stimulated emission depletion microscopy, photoactivated localization microscopy, stochastic optical reconstruction microscopy) approaches. However, they either operate in the challenging near-field mode or there is the need to label samples in biology. Recently, through manipulation of the diffraction of light with binary masks or gradient metasurfaces, some miniaturized and planar lenses have been reported with intriguing functionalities such as ultrahigh numerical aperture, large depth of focus, and subdiffraction-limit focusing in far-field, which provides a viable solution for the label-free superresolution imaging. Here, the recent advances in planar diffractive lenses (PDLs) are reviewed from a united theoretical account on diffraction-based focusing optics, and the underlying physics of nanofocusing via constructive or destructive interference is revealed. Various approaches of realizing PDLs are introduced in terms of their unique performances and interpreted by using optical aberration theory. Furthermore, a detailed tutorial about applying these planar lenses in nanoimaging is provided, followed by an outlook regarding future development toward practical applications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Doppler optical coherence microscopy and tomography applied to inner ear mechanics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Page, Scott; Freeman, Dennis M.; Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts

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-nanometermore » 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.« less

An introduction to optical super-resolution microscopy for the adventurous biologist

NASA Astrophysics Data System (ADS)

Vangindertael, J.; Camacho, R.; Sempels, W.; Mizuno, H.; Dedecker, P.; Janssen, K. P. F.

2018-04-01

Ever since the inception of light microscopy, the laws of physics have seemingly thwarted every attempt to visualize the processes of life at its most fundamental, sub-cellular, level. The diffraction limit has restricted our view to length scales well above 250 nm and in doing so, severely compromised our ability to gain true insights into many biological systems. Fortunately, continuous advancements in optics, electronics and mathematics have since provided the means to once again make physics work to our advantage. Even though some of the fundamental concepts enabling super-resolution light microscopy have been known for quite some time, practically feasible implementations have long remained elusive. It should therefore not come as a surprise that the 2014 Nobel Prize in Chemistry was awarded to the scientists who, each in their own way, contributed to transforming super-resolution microscopy from a technological tour de force to a staple of the biologist’s toolkit. By overcoming the diffraction barrier, light microscopy could once again be established as an indispensable tool in an age where the importance of understanding life at the molecular level cannot be overstated. This review strives to provide the aspiring life science researcher with an introduction to optical microscopy, starting from the fundamental concepts governing compound and fluorescent confocal microscopy to the current state-of-the-art of super-resolution microscopy techniques and their applications.

Quantitative analysis with advanced compensated polarized light microscopy on wavelength dependence of linear birefringence of single crystals causing arthritis

NASA Astrophysics Data System (ADS)

Takanabe, Akifumi; Tanaka, Masahito; Taniguchi, Atsuo; Yamanaka, Hisashi; Asahi, Toru

2014-07-01

To improve our ability to identify single crystals causing arthritis, we have developed a practical measurement system of polarized light microscopy called advanced compensated polarized light microscopy (A-CPLM). The A-CPLM system is constructed by employing a conventional phase retardation plate, an optical fibre and a charge-coupled device spectrometer in a polarized light microscope. We applied the A-CPLM system to measure linear birefringence (LB) in the visible region, which is an optical anisotropic property, for tiny single crystals causing arthritis, i.e. monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD). The A-CPLM system performance was evaluated by comparing the obtained experimental data using the A-CPLM system with (i) literature data for a standard sample, MgF2, and (ii) experimental data obtained using an established optical method, high-accuracy universal polarimeter, for the MSUM. The A-CPLM system was found to be applicable for measuring the LB spectra of the single crystals of MSUM and CPPD, which cause arthritis, in the visible regions. We quantitatively reveal the large difference in LB between MSUM and CPPD crystals. These results demonstrate the usefulness of the A-CPLM system for distinguishing the crystals causing arthritis.

Optical measurement of arterial mechanical properties: from atherosclerotic plaque initiation to rupture

NASA Astrophysics Data System (ADS)

Nadkarni, Seemantini K.

2013-12-01

During the pathogenesis of coronary atherosclerosis, from lesion initiation to rupture, arterial mechanical properties are altered by a number of cellular, molecular, and hemodynamic processes. There is growing recognition that mechanical factors may actively drive vascular cell signaling and regulate atherosclerosis disease progression. In advanced plaques, the mechanical properties of the atheroma influence stress distributions in the fibrous cap and mediate plaque rupture resulting in acute coronary events. This review paper explores current optical technologies that provide information on the mechanical properties of arterial tissue to advance our understanding of the mechanical factors involved in atherosclerosis development leading to plaque rupture. The optical approaches discussed include optical microrheology and traction force microscopy that probe the mechanical behavior of single cell and extracellular matrix components, and intravascular imaging modalities including laser speckle rheology, optical coherence elastography, and polarization-sensitive optical coherence tomography to measure the mechanical properties of advanced coronary lesions. Given the wealth of information that these techniques can provide, optical imaging modalities are poised to play an increasingly significant role in elucidating the mechanical aspects of coronary atherosclerosis in the future.

Advances in Light Microscopy for Neuroscience

PubMed Central

Wilt, Brian A.; Burns, Laurie D.; Ho, Eric Tatt Wei; Ghosh, Kunal K.; Mukamel, Eran A.

2010-01-01

Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists. PMID:19555292

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

Here, there and everywhere: The art and science of optics at work

NASA Astrophysics Data System (ADS)

Ambrosini, Dario; Ferraro, Pietro

2018-05-01

Optics, the ancient science of vision and light [1-5] can look forward to a "bright" future [6,7], as its applications are now ubiquitous in fields as diverse as science, engineering, technology, medicine and everyday life. Optical methods play a crucial and often revolutionary role in non-destructive testing, biomedical applications, microscopy, cultural heritage protection, advanced imaging in medicine, development of self-driving cars, astronomy, remote sensing, and manufacturing to cite a few examples.

Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, P.-F.; Yeh, Alvin T.; Bayless, Kayla J.

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 observedmore » 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.« less

Photoacoustic microscopy and computed tomography: from bench to bedside

PubMed Central

Wang, Lihong V.; Gao, Liang

2014-01-01

Photoacoustic imaging (PAI) of biological tissue has seen immense growth in the past decade, providing unprecedented spatial resolution and functional information at depths in the optical diffusive regime. PAI uniquely combines the advantages of optical excitation and acoustic detection. The hybrid imaging modality features high sensitivity to optical absorption and wide scalability of spatial resolution with the desired imaging depth. Here we first summarize the fundamental principles underpinning the technology, then highlight its practical implementation, and finally discuss recent advances towards clinical translation. PMID:24905877

Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections

NASA Astrophysics Data System (ADS)

DeArmond, Fredrick Michael

As optical microscopy techniques continue to improve, most notably the development of super-resolution optical microscopy which garnered the Nobel Prize in Chemistry in 2014, renewed emphasis has been placed on the development and use of fluorescence microscopy techniques. Of particular note is a renewed interest in multiphoton excitation due to a number of inherent properties of the technique including simplified optical filtering, increased sample penetration, and inherently confocal operation. With this renewed interest in multiphoton fluorescence microscopy, comes an increased demand for robust non-linear fluorescent markers, and characterization of the associated tool set. These factors have led to an experimental setup to allow a systematized approach for identifying and characterizing properties of fluorescent probes in the hopes that the tool set will provide researchers with additional information to guide their efforts in developing novel fluorophores suitable for use in advanced optical microscopy techniques as well as identifying trends for their synthesis. Hardware was setup around a software control system previously developed. Three experimental tool sets were set up, characterized, and applied over the course of this work. These tools include scanning multiphoton fluorescence microscope with single molecule sensitivity, an interferometric autocorrelator for precise determination of the bandwidth and pulse width of the ultrafast Titanium Sapphire excitation source, and a simplified fluorescence microscope for the measurement of two-photon absorption cross sections. Resulting values for two-photon absorption cross sections and two-photon absorption action cross sections for two standardized fluorophores, four commercially available fluorophores, and ten novel fluorophores are presented as well as absorption and emission spectra.

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection

PubMed Central

Brown, Koshonna; Thurn, Ted; Xin, Lun; Liu, William; Bazak, Remon; Chen, Si; Lai, Barry; Vogt, Stefan; Jacobsen, Chris; Paunesku, Tatjana; Woloschak, Gayle E.

2018-01-01

Titanium dioxide (TiO2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here. PMID:29541425

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection.

PubMed

Brown, Koshonna; Thurn, Ted; Xin, Lun; Liu, William; Bazak, Remon; Chen, Si; Lai, Barry; Vogt, Stefan; Jacobsen, Chris; Paunesku, Tatjana; Woloschak, Gayle E

2018-01-01

Titanium dioxide (TiO 2 ) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.

Nonlinear plasmonic imaging techniques and their biological applications

NASA Astrophysics Data System (ADS)

Deka, Gitanjal; Sun, Chi-Kuang; Fujita, Katsumasa; Chu, Shi-Wei

2017-01-01

Nonlinear optics, when combined with microscopy, is known to provide advantages including novel contrast, deep tissue observation, and minimal invasiveness. In addition, special nonlinearities, such as switch on/off and saturation, can enhance the spatial resolution below the diffraction limit, revolutionizing the field of optical microscopy. These nonlinear imaging techniques are extremely useful for biological studies on various scales from molecules to cells to tissues. Nevertheless, in most cases, nonlinear optical interaction requires strong illumination, typically at least gigawatts per square centimeter intensity. Such strong illumination can cause significant phototoxicity or even photodamage to fragile biological samples. Therefore, it is highly desirable to find mechanisms that allow the reduction of illumination intensity. Surface plasmon, which is the collective oscillation of electrons in metal under light excitation, is capable of significantly enhancing the local field around the metal nanostructures and thus boosting up the efficiency of nonlinear optical interactions of the surrounding materials or of the metal itself. In this mini-review, we discuss the recent progress of plasmonics in nonlinear optical microscopy with a special focus on biological applications. The advancement of nonlinear imaging modalities (including incoherent/coherent Raman scattering, two/three-photon luminescence, and second/third harmonic generations that have been amalgamated with plasmonics), as well as the novel subdiffraction limit imaging techniques based on nonlinear behaviors of plasmonic scattering, is addressed.

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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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-fieldmore » 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 find the 3D orientation of stationary metallic anisotropic nanoparticles utilizing only long-axis SPR enhancement. The polarization direction of the illuminating light was rotated causing the relative intensity of p-polarized and s-polarized light within the evanescent field to change. The interaction of the evanescent field with the particles is dependent on the orientation of the particle producing an intensity curve. This curve and the in-plane angle can be compared with simulations to accurately determine the 3D orientation. Differential interference contrast (DIC) microscopy is another non-invasive far-field technique based upon interferometry that does not rely on staining or other contrast enhancing techniques. In addition, high numerical aperture condensers and objectives can be used to give a very narrow depth of field allowing for the optical tomography of samples, which makes it an ideal candidate to study biological systems. DIC microscopy has also proven itself in determining the orientation of gold nanorods in both engineered environments and within cells. Many types of nanoparticles and nanostructures have been synthesized using lithographic techniques on silicon wafer substrates. Traditionally, reflective mode DIC microscopes have been developed and applied to the topographical study of reflective substrates and the imaging of chips on silicon wafers. Herein, a laser-illuminated reflected-mode DIC was developed for studying nanoparticles on reflective surfaces.« less

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 find the 3D orientation of stationary metallic anisotropic nanoparticles utilizing only long-axis SPR enhancement. The polarization direction of the illuminating light was rotated causing the relative intensity of p-polarized and s-polarized light within the evanescent field to change. The interaction of the evanescent field with the particles is dependent on the orientation of the particle producing an intensity curve. This curve and the in-plane angle can be compared with simulations to accurately determine the 3D orientation. Differential interference contrast (DIC) microscopy is another non-invasive far-field technique based upon interferometry that does not rely on staining or other contrast enhancing techniques. In addition, high numerical aperture condensers and objectives can be used to give a very narrow depth of field allowing for the optical tomography of samples, which makes it an ideal candidate to study biological systems. DIC microscopy has also proven itself in determining the orientation of gold nanorods in both engineered environments and within cells. Many types of nanoparticles and nanostructures have been synthesized using lithographic techniques on silicon wafer substrates. Traditionally, reflective mode DIC microscopes have been developed and applied to the topographical study of reflective substrates and the imaging of chips on silicon wafers. Herein, a laser-illuminated reflected-mode DIC was developed for studying nanoparticles on reflective surfaces.

Automatic and adaptive heterogeneous refractive index compensation for light-sheet microscopy.

PubMed

Ryan, Duncan P; Gould, Elizabeth A; Seedorf, Gregory J; Masihzadeh, Omid; Abman, Steven H; Vijayaraghavan, Sukumar; Macklin, Wendy B; Restrepo, Diego; Shepherd, Douglas P

2017-09-20

Optical tissue clearing has revolutionized researchers' ability to perform fluorescent measurements of molecules, cells, and structures within intact tissue. One common complication to all optically cleared tissue is a spatially heterogeneous refractive index, leading to light scattering and first-order defocus. We designed C-DSLM (cleared tissue digital scanned light-sheet microscopy) as a low-cost method intended to automatically generate in-focus images of cleared tissue. We demonstrate the flexibility and power of C-DSLM by quantifying fluorescent features in tissue from multiple animal models using refractive index matched and mismatched microscope objectives. This includes a unique measurement of myelin tracks within intact tissue using an endogenous fluorescent reporter where typical clearing approaches render such structures difficult to image. For all measurements, we provide independent verification using standard serial tissue sectioning and quantification methods. Paired with advancements in volumetric image processing, C-DSLM provides a robust methodology to quantify sub-micron features within large tissue sections.Optical clearing of tissue has enabled optical imaging deeper into tissue due to significantly reduced light scattering. Here, Ryan et al. tackle first-order defocus, an artefact of a non-uniform refractive index, extending light-sheet microscopy to partially cleared samples.

Biophotonic endoscopy: a review of clinical research techniques for optical imaging and sensing of early gastrointestinal cancer

PubMed Central

Coda, Sergio; Siersema, Peter D.; Stamp, Gordon W. H.; Thillainayagam, Andrew V.

2015-01-01

Detection, characterization, and staging constitute the fundamental elements in the endoscopic diagnosis of gastrointestinal diseases, but histology still remains the diagnostic gold standard. New developments in endoscopic techniques may challenge histopathology in the near future. An ideal endoscopic technique should combine a wide-field, “red flag” screening technique with an optical contrast or microscopy method for characterization and staging, all simultaneously available during the procedure. In theory, biophotonic advances have the potential to unite these elements to allow in vivo “optical biopsy.” These techniques may ultimately offer the potential to increase the rates of detection of high risk lesions and the ability to target biopsies and resections, and so reduce the need for biopsy, costs, and uncertainty for patients. However, their utility and sensitivity in clinical practice must be evaluated against those of conventional histopathology. This review describes some of the most recent applications of biophotonics in endoscopic optical imaging and metrology, along with their fundamental principles and the clinical experience that has been acquired in their deployment as tools for the endoscopist. Particular emphasis has been placed on translational label-free optical techniques, such as fluorescence spectroscopy, fluorescence lifetime imaging microscopy (FLIM), two-photon and multi-photon microscopy, second harmonic generation (SHG) and third harmonic generation (THG) imaging, optical coherence tomography (OCT), diffuse reflectance, Raman spectroscopy, and molecular imaging. PMID:26528489

Attomicroscopy: from femtosecond to attosecond electron microscopy

NASA Astrophysics Data System (ADS)

Hassan, Mohammed Th

2018-02-01

In the last decade, the development of ultrafast electron diffraction (UED) and microscopy (UEM) have enabled the imaging of atomic motion in real time and space. These pivotal table-top tools opened the door for a vast range of applications in different areas of science spanning chemistry, physics, materials science, and biology. We first discuss the basic principles and recent advancements, including some of the important applications, of both UED and UEM. Then, we discuss the recent advances in the field that have enhanced the spatial and temporal resolutions, where the latter, is however, still limited to a few hundreds of femtoseconds, preventing the imaging of ultrafast dynamics of matter lasting few tens of femtoseconds. Then, we present our new optical gating approach for generating an isolated 30 fs electron pulse with sufficient intensity to attain a temporal resolution on the same time scale. This achievement allows, for the first time, imaging the electron dynamics of matter. Finally, we demonstrate the feasibility of the optical gating approach to generate an isolated attosecond electron pulse, utilizing our recently demonstrated optical attosecond laser pulse, which paves the way for establishing the field of ‘Attomicroscopy’, ultimately enabling us to image the electron motion in action.

Intracellular in situ labeling of TiO 2 nanoparticles for fluorescence microscopy detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Koshonna; Thurn, Ted; Xin, Lun

Titanium dioxide (TiO 2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. In this paper, we describe two in situ posttreatmentmore » labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Finally and therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.« less

Intracellular in situ labeling of TiO 2 nanoparticles for fluorescence microscopy detection

DOE PAGES

Brown, Koshonna; Thurn, Ted; Xin, Lun; ...

2017-07-19

Titanium dioxide (TiO 2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. In this paper, we describe two in situ posttreatmentmore » labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Finally and therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.« less

Optical Diagnostics in Medicine

NASA Astrophysics Data System (ADS)

Iftimia, Nicusor

2003-03-01

Light has a unique potential for non-invasive tissue diagnosis. The relatively short wavelength of light allows imaging of tissue at the resolution of histopathology. While strong multiple scattering of light in tissue makes attainment of this resolution difficult for thick tissues, most pathology emanates from epithelial surfaces. Therefore, high-resolution diagnosis of many important diseases may be achieved by transmitting light to the surface of interest. The recent fiber-optic implementation of technologies that reject multiple scattering, such as confocal microscopy and optical low coherence interferometry, have brought us one step closer to realizing non-invasive imaging of architectural and cellular features of tissue. Optical coherence tomography (OCT) can produce high-resolution cross-sectional images of biological structures. Clinical OCT studies conducted in the gastrointestinal tract and cardiovascular system have shown that OCT is capable of providing images of the architectural (> 20 µm) microanatomy of a variety of epithelial tissues, including the layered structure of squamous epithelium and arterial vessels. Fine Needle Aspiration- Low Coherence Interferometry (FNA-LCI) is another optical diagnostics technique, which is a suitable solution to increase the effectiveness of the FNA procedures. LCI is capable of measuring depth resolved (axial, z) tissue structure, birefringence, flow (Doppler shift), and spectra at a resolution of several microns. Since LCI systems are fiber-optic based, LCI probes may easily fit within the bore of a fine gauge needle, allowing diagnostic information to be obtained directly from the FNA biopsy site. Fiber optic spectrally encoded confocal microscopy (SECM) is a new confocal microscopy method, which eliminates the need for rapid beam scanning within the optical probe. This advance enables confocal microscopy to be performed through small diameter probes and will allow assessment of internal human tissues in vivo at the cellular level. A detailed description of several fiber optics based systems for early diseases diagnosis, as well as preliminary clinic results, will be presented.

Mueller matrix signature in advanced fluorescence microscopy imaging

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

Far-field optical imaging with subdiffraction resolution enabled by nonlinear saturation absorption

NASA Astrophysics Data System (ADS)

Ding, Chenliang; Wei, Jingsong

2016-01-01

The resolution of far-field optical imaging is required to improve beyond the Abbe limit to the subdiffraction or even the nanoscale. In this work, inspired by scanning electronic microscopy (SEM) imaging, in which carbon (or Au) thin films are usually required to be coated on the sample surface before imaging to remove the charging effect while imaging by electrons. We propose a saturation-absorption-induced far-field super-resolution optical imaging method (SAI-SRIM). In the SAI-SRIM, the carbon (or Au) layers in SEM imaging are replaced by nonlinear-saturation-absorption (NSA) thin films, which are directly coated onto the sample surfaces using advanced thin film deposition techniques. The surface fluctuant morphologies are replicated to the NSA thin films, accordingly. The coated sample surfaces are then imaged using conventional laser scanning microscopy. Consequently, the imaging resolution is greatly improved, and subdiffraction-resolved optical images are obtained theoretically and experimentally. The SAI-SRIM provides an effective and easy way to achieve far-field super-resolution optical imaging for sample surfaces with geometric fluctuant morphology characteristics.

Light-neuron interactions: key to understanding the brain

NASA Astrophysics Data System (ADS)

Go, Mary Ann; Daria, Vincent R.

2017-02-01

In recent years, advances in light-based technology have driven an ongoing optical revolution in neuroscience. Synergistic technologies in laser microscopy, molecular biology, organic and synthetic chemistry, genetic engineering and materials science have allowed light to overcome the limitations of and to replace many conventional tools used by physiologists to record from and to manipulate single cells or whole cellular networks. Here we review the different optical techniques for stimulating neurons, influencing neuronal growth, manipulating neuronal structures and neurosurgery.

Microscopy imaging device with advanced imaging properties

DOEpatents

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

2015-11-24

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

Microscopy imaging device with advanced imaging properties

DOEpatents

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

2016-10-25

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

Microscopy imaging device with advanced imaging properties

DOEpatents

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

2016-11-22

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

Microscopy imaging device with advanced imaging properties

DOEpatents

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

2017-04-25

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

The emergence of optical elastography in biomedicine

NASA Astrophysics Data System (ADS)

Kennedy, Brendan F.; Wijesinghe, Philip; Sampson, David D.

2017-04-01

Optical elastography, the use of optics to characterize and map the mechanical properties of biological tissue, involves measuring the deformation of tissue in response to a load. Such measurements may be used to form an image of a mechanical property, often elastic modulus, with the resulting mechanical contrast complementary to the more familiar optical contrast. Optical elastography is experiencing new impetus in response to developments in the closely related fields of cell mechanics and medical imaging, aided by advances in photonics technology, and through probing the microscale between that of cells and whole tissues. Two techniques -- optical coherence elastography and Brillouin microscopy -- have recently shown particular promise for medical applications, such as in ophthalmology and oncology, and as new techniques in cell mechanics.

Hemodynamic flow visualization of early embryonic great vessels using μPIV.

PubMed

Goktas, Selda; Chen, Chia-Yuan; Kowalski, William J; Pekkan, Kerem

2015-01-01

Microparticle image velocimetry (μPIV) is an evolving quantitative methodology to closely and accurately monitor the cardiac flow dynamics and mechanotransduction during vascular morphogenesis. While PIV technique has a long history, contemporary developments in advanced microscopy have significantly expanded its power. This chapter includes three new methods for μPIV acquisition in selected embryonic structures achieved through advanced optical imaging: (1) high-speed confocal scanning of transgenic zebrafish embryos, where the transgenic erythrocytes act as the tracing particles; (2) microinjection of artificial seeding particles in chick embryos visualized with stereomicroscopy; and (3) real-time, time-resolved optical coherence tomography acquisition of vitelline vessel flow profiles in chick embryos, tracking the erythrocytes.

A hard X-ray nanoprobe beamline for nanoscale microscopy.

PubMed

Winarski, Robert P; Holt, Martin V; Rose, Volker; Fuesz, Peter; Carbaugh, Dean; Benson, Christa; Shu, Deming; Kline, David; Stephenson, G Brian; McNulty, Ian; Maser, Jörg

2012-11-01

The Hard X-ray Nanoprobe Beamline (or Nanoprobe Beamline) is an X-ray microscopy facility incorporating diffraction, fluorescence and full-field imaging capabilities designed and operated by the Center for Nanoscale Materials and the Advanced Photon Source at Sector 26 of the Advanced Photon Source at Argonne National Laboratory. This facility was constructed to probe the nanoscale structure of biological, environmental and material sciences samples. The beamline provides intense focused X-rays to the Hard X-ray Nanoprobe (or Nanoprobe) which incorporates Fresnel zone plate optics and a precision laser sensing and control system. The beamline operates over X-ray energies from 3 to 30 keV, enabling studies of most elements in the periodic table, with a particular emphasis on imaging transition metals.

Orientational imaging of a single plasmonic nanoparticle using dark-field hyperspectral imaging

NASA Astrophysics Data System (ADS)

Mehta, Nishir; Mahigir, Amirreza; Veronis, Georgios; Gartia, Manas Ranjan

2017-08-01

Orientation of plasmonic nanostructures is an important feature in many nanoscale applications such as catalyst, biosensors DNA interactions, protein detections, hotspot of surface enhanced Raman spectroscopy (SERS), and fluorescence resonant energy transfer (FRET) experiments. However, due to diffraction limit, it is challenging to obtain the exact orientation of the nanostructure using standard optical microscope. Hyperspectral Imaging Microscopy is a state-of-the-art visualization technology that combines modern optics with hyperspectral imaging and computer system to provide the identification and quantitative spectral analysis of nano- and microscale structures. In this work, initially we use transmitted dark field imaging technique to locate single nanoparticle on a glass substrate. Then we employ hyperspectral imaging technique at the same spot to investigate orientation of single nanoparticle. No special tagging or staining of nanoparticle has been done, as more likely required in traditional microscopy techniques. Different orientations have been identified by carefully understanding and calibrating shift in spectral response from each different orientations of similar sized nanoparticles. Wavelengths recorded are between 300 nm to 900 nm. The orientations measured by hyperspectral microscopy was validated using finite difference time domain (FDTD) electrodynamics calculations and scanning electron microscopy (SEM) analysis. The combination of high resolution nanometer-scale imaging techniques and the modern numerical modeling capacities thus enables a meaningful advance in our knowledge of manipulating and fabricating shaped nanostructures. This work will advance our understanding of the behavior of small nanoparticle clusters useful for sensing, nanomedicine, and surface sciences.

Diffraction and microscopy with attosecond electron pulse trains

NASA Astrophysics Data System (ADS)

Morimoto, Yuya; Baum, Peter

2018-03-01

Attosecond spectroscopy1-7 can resolve electronic processes directly in time, but a movie-like space-time recording is impeded by the too long wavelength ( 100 times larger than atomic distances) or the source-sample entanglement in re-collision techniques8-11. Here we advance attosecond metrology to picometre wavelength and sub-atomic resolution by using free-space electrons instead of higher-harmonic photons1-7 or re-colliding wavepackets8-11. A beam of 70-keV electrons at 4.5-pm de Broglie wavelength is modulated by the electric field of laser cycles into a sequence of electron pulses with sub-optical-cycle duration. Time-resolved diffraction from crystalline silicon reveals a < 10-as delay of Bragg emission and demonstrates the possibility of analytic attosecond-ångström diffraction. Real-space electron microscopy visualizes with sub-light-cycle resolution how an optical wave propagates in space and time. This unification of attosecond science with electron microscopy and diffraction enables space-time imaging of light-driven processes in the entire range of sample morphologies that electron microscopy can access.

Advanced Fiber-optic Monitoring System for Space-flight Applications

NASA Technical Reports Server (NTRS)

Hull, M. S.; VanTassell, R. L.; Pennington, C. D.; Roman, M.

2005-01-01

Researchers at Luna Innovations Inc. and the National Aeronautic and Space Administration s Marshall Space Flight Center (NASA MSFC) have developed an integrated fiber-optic sensor system for real-time monitoring of chemical contaminants and whole-cell bacterial pathogens in water. The system integrates interferometric and evanescent-wave optical fiber-based sensing methodologies with atomic force microscopy (AFM) and long-period grating (LPG) technology to provide versatile measurement capability for both micro- and nano-scale analytes. Sensors can be multiplexed in an array format and embedded in a totally self-contained laboratory card for use with an automated microfluidics platform.

Photoluminescence analysis of self induced planer alignment in azo dye dispersed nematic liquid crystal complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Rishi, E-mail: kkraina@gmail.com; Sood, Srishti, E-mail: kkraina@gmail.com; Raina, K. K., E-mail: kkraina@gmail.com

2014-04-24

We have developed azo dye doped nematic liquid crystal complex for advanced photonic liquid crystal display technology aspects. Disperse orange azo dye self introduced planer alignment in the nematic liquid crystal without any surface anchoring treatment. Planer alignment was characterized by optical polarizing microscopy. The electro-optical switching response of dye disperse planer aligned nematic cell was investigated as a function of applied voltage with the help of photoluminescence spectrophotometer for the tuning of photoluminescence contrast.

3D wide field-of-view Gabor-domain optical coherence microscopy advancing real-time in-vivo imaging and metrology

NASA Astrophysics Data System (ADS)

Canavesi, Cristina; Cogliati, Andrea; Hayes, Adam; Tankam, Patrice; Santhanam, Anand; Rolland, Jannick P.

2017-02-01

Real-time volumetric high-definition wide-field-of-view in-vivo cellular imaging requires micron-scale resolution in 3D. Compactness of the handheld device and distortion-free images with cellular resolution are also critically required for onsite use in clinical applications. By integrating a custom liquid lens-based microscope and a dual-axis MEMS scanner in a compact handheld probe, Gabor-domain optical coherence microscopy (GD-OCM) breaks the lateral resolution limit of optical coherence tomography through depth, overcoming the tradeoff between numerical aperture and depth of focus, enabling advances in biotechnology. Furthermore, distortion-free imaging with no post-processing is achieved with a compact, lightweight handheld MEMS scanner that obtained a 12-fold reduction in volume and 17-fold reduction in weight over a previous dual-mirror galvanometer-based scanner. Approaching the holy grail of medical imaging - noninvasive real-time imaging with histologic resolution - GD-OCM demonstrates invariant resolution of 2 μm throughout a volume of 1 x 1 x 0.6 mm3, acquired and visualized in less than 2 minutes with parallel processing on graphics processing units. Results on the metrology of manufactured materials and imaging of human tissue with GD-OCM are presented.

Neurosurgical confocal endomicroscopy: A review of contrast agents, confocal systems, and future imaging modalities

PubMed Central

Zehri, Aqib H.; Ramey, Wyatt; Georges, Joseph F.; Mooney, Michael A.; Martirosyan, Nikolay L.; Preul, Mark C.; Nakaji, Peter

2014-01-01

Background: The clinical application of fluorescent contrast agents (fluorescein, indocyanine green, and aminolevulinic acid) with intraoperative microscopy has led to advances in intraoperative brain tumor imaging. Their properties, mechanism of action, history of use, and safety are analyzed in this report along with a review of current laser scanning confocal endomicroscopy systems. Additional imaging modalities with potential neurosurgical utility are also analyzed. Methods: A comprehensive literature search was performed utilizing PubMed and key words: In vivo confocal microscopy, confocal endomicroscopy, fluorescence imaging, in vivo diagnostics/neoplasm, in vivo molecular imaging, and optical imaging. Articles were reviewed that discussed clinically available fluorophores in neurosurgery, confocal endomicroscopy instrumentation, confocal microscopy systems, and intraoperative cancer diagnostics. Results: Current clinically available fluorescent contrast agents have specific properties that provide microscopic delineation of tumors when imaged with laser scanning confocal endomicroscopes. Other imaging modalities such as coherent anti-Stokes Raman scattering (CARS) microscopy, confocal reflectance microscopy, fluorescent lifetime imaging (FLIM), two-photon microscopy, and second harmonic generation may also have potential in neurosurgical applications. Conclusion: In addition to guiding tumor resection, intraoperative fluorescence and microscopy have the potential to facilitate tumor identification and complement frozen section analysis during surgery by providing real-time histological assessment. Further research, including clinical trials, is necessary to test the efficacy of fluorescent contrast agents and optical imaging instrumentation in order to establish their role in neurosurgery. PMID:24872922

Seeing and believing: recent advances in imaging cell-cell interactions

PubMed Central

Yap, Alpha S.; Michael, Magdalene; Parton, Robert G.

2015-01-01

Advances in cell and developmental biology have often been closely linked to advances in our ability to visualize structure and function at many length and time scales. In this review, we discuss how new imaging technologies and new reagents have provided novel insights into the biology of cadherin-based cell-cell junctions. We focus on three developments: the application of super-resolution optical technologies to characterize the nanoscale organization of cadherins at cell-cell contacts, new approaches to interrogate the mechanical forces that act upon junctions, and advances in electron microscopy which have the potential to transform our understanding of cell-cell junctions. PMID:26543555

Seeing and believing: recent advances in imaging cell-cell interactions.

PubMed

Yap, Alpha S; Michael, Magdalene; Parton, Robert G

2015-01-01

Advances in cell and developmental biology have often been closely linked to advances in our ability to visualize structure and function at many length and time scales. In this review, we discuss how new imaging technologies and new reagents have provided novel insights into the biology of cadherin-based cell-cell junctions. We focus on three developments: the application of super-resolution optical technologies to characterize the nanoscale organization of cadherins at cell-cell contacts, new approaches to interrogate the mechanical forces that act upon junctions, and advances in electron microscopy which have the potential to transform our understanding of cell-cell junctions.

Advanced Wide-Field Interferometric Microscopy for Nanoparticle Sensing and Characterization

NASA Astrophysics Data System (ADS)

Avci, Oguzhan

Nanoparticles have a key role in today's biotechnological research owing to the rapid advancement of nanotechnology. While metallic, polymer, and semiconductor based artificial nanoparticles are widely used as labels or targeted drug delivery agents, labeled and label-free detection of natural nanoparticles promise new ways for viral diagnostics and therapeutic applications. The increasing impact of nanoparticles in bio- and nano-technology necessitates the development of advanced tools for their accurate detection and characterization. Optical microscopy techniques have been an essential part of research for visualizing micron-scale particles. However, when it comes to the visualization of individual nano-scale particles, they have shown inadequate success due to the resolution and visibility limitations. Interferometric microscopy techniques have gained significant attention for providing means to overcome the nanoparticle visibility issue that is often the limiting factor in the imaging techniques based solely on the scattered light. In this dissertation, we develop a rigorous physical model to simulate the single nanoparticle optical response in a common-path wide-field interferometric microscopy (WIM) system. While the fundamental elements of the model can be used to analyze nanoparticle response in any generic wide-field imaging systems, we focus on imaging with a layered substrate (common-path interferometer) where specular reflection of illumination provides the reference light for interferometry. A robust physical model is quintessential in realizing the full potential of an optical system, and throughout this dissertation, we make use of it to benchmark our experimental findings, investigate the utility of various optical configurations, reconstruct weakly scattering nanoparticle images, as well as to characterize and discriminate interferometric nanoparticle responses. This study investigates the integration of advanced optical schemes in WIM with two main goals in mind: (i) increasing the visibility of low-index nanoscale particles via pupil function engineering, pushing the limit of sensitivity; (ii) improving the resolution of sub-diffraction-limited, low-index particle images in WIM via reconstruction strategies for shape and orientation information. We successfully demonstrate an overall ten-fold improvement in the visibility of the low-index sub-wavelength nanoparticles as well as up to two-fold extended spatial resolution of the interference-enhanced nanoparticle images. We also systematically examine the key factors that determine the signal in WIM. These factors include the particle type, size, layered substrate design, defocus and nanoparticle polarizability. We use the physical model to demonstrate how these factors determine the signal levels, and demonstrate how the layered substrate can be designed to optimize the overall signal, while defocus scan can be used to maximize it, as well as its signature can be utilized for particle discrimination purposes for both dielectric particles and resonant metallic particles. We introduce a machine learning based particle characterization algorithm that relies on supervised learning from model. The particle characterization is limited to discrimination based on nanosphere size and type in the scope of this dissertation.

On the chemical homogeneity of In xGa 1–xN alloys – Electron microscopy at the edge of technical limits

DOE PAGES

Specht, Petra; Kisielowski, Christian

2016-08-30

Ternary In xGa 1–xN alloys became technologically attractive when p-doping was achieved to produce blue and green light emitting diodes (LED)s. Starting in the mid 1990th, investigations of their chemical homogeneity were driven by the need to understand carrier recombination mechanisms in optical device structures to optimize their performance. Transmission electron microscopy (TEM) is the technique of choice to complement optical data evaluations, which suggests the coexistence of local carrier recombination mechanisms based on piezoelectric field effects and on indium clustering in the quantum wells of LEDs. We summarize the historic context of homogeneity investigations using electron microscopy techniques thatmore » can principally resolve the question of indium segregation and clustering in In xGa 1–xN alloys if optimal sample preparation and electron dose-controlled imaging techniques are employed together with advanced data evaluation.« less

Imaging genes, chromosomes, and nuclear structures using laser-scanning confocal microscopy

NASA Astrophysics Data System (ADS)

Ballard, Stephen G.

1990-08-01

For 350 years, the optical microscope has had a powerful symbiotic relationship with biology. Until this century, optical microscopy was the only means of examining cellular structure; in return, biologists have contributed greatly to the evolution of microscope design and technique. Recent advances in the detection and processing of optical images, together with methods for labelling specific biological molecules, have brought about a resurgence in the application of optical microscopy to the biological sciences. One of the areas in which optical microscopy is breaking new ground is in elucidating the large scale organization of chromatin in chromosomes and cell nuclei. Nevertheless, imaging the contents of the cell nucleus is a difficult challenge for light microscopy, for two principal reasons. First, the dimensions of all but the largest nuclear structures (nucleoli, vacuoles) are close to or below the resolving power of far field optics. Second, the native optical contrast properties of many important chromatin structures (eg. chromosome domains, centromere regions) are very weak, or essentially zero. As an extreme example, individual genes probably have nothing to distinguish them other than their sequence of DNA bases, which cannot be directly visualized with any current form of microscopy. Similarly, the interphase nucleus shows no direct visible evidence of focal chromatin domains. Thus, imaging of such entities depends heavily on contrast enhancement methods. The most promising of these is labelling DNA in situ using sequence-specific probes that may be visualized using fluorescent dyes. We have applied this method to detecting individual genes in metaphase chromosomes and interphase nuclei, and to imaging a number of DNA-containing structures including chromosome domains, metaphase chromosomes and centromere regions. We have also demonstrated the applicability of in situ fluorescent labelling to detecting numerical and structural abnormalities both in condensed metaphase chromosomes and in interphase nuclei. The ability to image the loci of fluorescent-labelled gene probes hybridized to chromosomes and to interphase nuclei will play a major role in the mapping of the human genome. This presentation is an overview of our laboratory's efforts to use confocal imaging to address fundamental questions about the structure and organization of genes, chromosomes and cell nuclei, and to develop applications useful in clinical diagnosis of inherited diseases.

X-ray ptychography

NASA Astrophysics Data System (ADS)

Pfeiffer, Franz

2018-01-01

X-ray ptychographic microscopy combines the advantages of raster scanning X-ray microscopy with the more recently developed techniques of coherent diffraction imaging. It is limited neither by the fabricational challenges associated with X-ray optics nor by the requirements of isolated specimen preparation, and offers in principle wavelength-limited resolution, as well as stable access and solution to the phase problem. In this Review, we discuss the basic principles of X-ray ptychography and summarize the main milestones in the evolution of X-ray ptychographic microscopy and tomography over the past ten years, since its first demonstration with X-rays. We also highlight the potential for applications in the life and materials sciences, and discuss the latest advanced concepts and probable future developments.

Using Polarized Spectroscopy to Investigate Order in Thin-Films of Ionic Self-Assembled Materials Based on Azo-Dyes

PubMed Central

Ahmad, Mariam; Andersen, Frederik; Brend Bech, Ári; Bendixen, H. Krestian L.; Nawrocki, Patrick R.; Bloch, Anders J.; Bora, Ilkay; Bukhari, Tahreem A.; Bærentsen, Nicolai V.; Carstensen, Jens; Chima, Smeeah; Colberg, Helene; Dahm, Rasmus T.; Daniels, Joshua A.; Dinckan, Nermin; El Idrissi, Mohamed; Erlandsen, Ricci; Førster, Marc; Ghauri, Yasmin; Gold, Mikkel; Hansen, Andreas; Hansen, Kenn; Helmsøe-Zinck, Mathias; Henriksen, Mathias; Hoffmann, Sophus V.; Hyllested, Louise O. H.; Jensen, Casper; Kallenbach, Amalie S.; Kaur, Kirandip; Khan, Suheb R.; Kjær, Emil T. S.; Kristiansen, Bjørn; Langvad, Sylvester; Lund, Philip M.; Munk, Chastine F.; Møller, Theis; Nehme, Ola M. Z.; Nejrup, Mathilde Rove; Nexø, Louise; Nielsen, Simon Skødt Holm; Niemeier, Nicolai; Nikolajsen, Lasse V.; Nøhr, Peter C. T.; Skaarup Ovesen, Jacob; Paustian, Lucas; Pedersen, Adam S.; Petersen, Mathias K.; Poulsen, Camilla M.; Praeger-Jahnsen, Louis; Qureshi, L. Sonia; Schiermacher, Louise S.; Simris, Martin B.; Smith, Gorm; Smith, Heidi N.; Sonne, Alexander K.; Zenulovic, Marko R.; Winther Sørensen, Alma; Vogt, Emil; Væring, Andreas; Westermann, Jonas; Özcan, Sevin B.

2018-01-01

Three series of ionic self-assembled materials based on anionic azo-dyes and cationic benzalkonium surfactants were synthesized and thin films were prepared by spin-casting. These thin films appear isotropic when investigated with polarized optical microscopy, although they are highly anisotropic. Here, three series of homologous materials were studied to rationalize this observation. Investigating thin films of ordered molecular materials relies to a large extent on advanced experimental methods and large research infrastructure. A statement that in particular is true for thin films with nanoscopic order, where X-ray reflectometry, X-ray and neutron scattering, electron microscopy and atom force microscopy (AFM) has to be used to elucidate film morphology and the underlying molecular structure. Here, the thin films were investigated using AFM, optical microscopy and polarized absorption spectroscopy. It was shown that by using numerical method for treating the polarized absorption spectroscopy data, the molecular structure can be elucidated. Further, it was shown that polarized optical spectroscopy is a general tool that allows determination of the molecular order in thin films. Finally, it was found that full control of thermal history and rigorous control of the ionic self-assembly conditions are required to reproducibly make these materials of high nanoscopic order. Similarly, the conditions for spin-casting are shown to be determining for the overall thin film morphology, while molecular order is maintained. PMID:29462883

Using Polarized Spectroscopy to Investigate Order in Thin-Films of Ionic Self-Assembled Materials Based on Azo-Dyes.

PubMed

Kühnel, Miguel R Carro-Temboury Martin; Ahmad, Mariam; Andersen, Frederik; Bech, Ári Brend; Bendixen, H Krestian L; Nawrocki, Patrick R; Bloch, Anders J; Bora, Ilkay; Bukhari, Tahreem A; Bærentsen, Nicolai V; Carstensen, Jens; Chima, Smeeah; Colberg, Helene; Dahm, Rasmus T; Daniels, Joshua A; Dinckan, Nermin; Idrissi, Mohamed El; Erlandsen, Ricci; Førster, Marc; Ghauri, Yasmin; Gold, Mikkel; Hansen, Andreas; Hansen, Kenn; Helmsøe-Zinck, Mathias; Henriksen, Mathias; Hoffmann, Sophus V; Hyllested, Louise O H; Jensen, Casper; Kallenbach, Amalie S; Kaur, Kirandip; Khan, Suheb R; Kjær, Emil T S; Kristiansen, Bjørn; Langvad, Sylvester; Lund, Philip M; Munk, Chastine F; Møller, Theis; Nehme, Ola M Z; Nejrup, Mathilde Rove; Nexø, Louise; Nielsen, Simon Skødt Holm; Niemeier, Nicolai; Nikolajsen, Lasse V; Nøhr, Peter C T; Orlowski, Dominik B; Overgaard, Marc; Ovesen, Jacob Skaarup; Paustian, Lucas; Pedersen, Adam S; Petersen, Mathias K; Poulsen, Camilla M; Praeger-Jahnsen, Louis; Qureshi, L Sonia; Ree, Nicolai; Schiermacher, Louise S; Simris, Martin B; Smith, Gorm; Smith, Heidi N; Sonne, Alexander K; Zenulovic, Marko R; Sørensen, Alma Winther; Sørensen, Karina; Vogt, Emil; Væring, Andreas; Westermann, Jonas; Özcan, Sevin B; Sørensen, Thomas Just

2018-02-15

Three series of ionic self-assembled materials based on anionic azo-dyes and cationic benzalkonium surfactants were synthesized and thin films were prepared by spin-casting. These thin films appear isotropic when investigated with polarized optical microscopy, although they are highly anisotropic. Here, three series of homologous materials were studied to rationalize this observation. Investigating thin films of ordered molecular materials relies to a large extent on advanced experimental methods and large research infrastructure. A statement that in particular is true for thin films with nanoscopic order, where X-ray reflectometry, X-ray and neutron scattering, electron microscopy and atom force microscopy (AFM) has to be used to elucidate film morphology and the underlying molecular structure. Here, the thin films were investigated using AFM, optical microscopy and polarized absorption spectroscopy. It was shown that by using numerical method for treating the polarized absorption spectroscopy data, the molecular structure can be elucidated. Further, it was shown that polarized optical spectroscopy is a general tool that allows determination of the molecular order in thin films. Finally, it was found that full control of thermal history and rigorous control of the ionic self-assembly conditions are required to reproducibly make these materials of high nanoscopic order. Similarly, the conditions for spin-casting are shown to be determining for the overall thin film morphology, while molecular order is maintained.

Perspectives on in situ electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Haimei; Zhu, Yimei

In situ transmission electron microscopy (TEM) with the ability to reveal materials dynamic processes with high spatial and temporal resolution has attracted significant interest. The recent advances in in situ methods, including liquid and gas sample environment, pump-probe ultrafast microscopy, nanomechanics and ferroelectric domain switching the aberration corrected electron optics as well as fast electron detector has opened new opportunities to extend the impact of in situ TEM in broad areas of research ranging from materials science to chemistry, physics and biology. Here in this paper, we highlight the development of liquid environment electron microscopy and its applications in themore » study of colloidal nanoparticle growth, electrochemical processes and others; in situ study of topological vortices in ferroelectric and ferromagnetic materials. At the end, perspectives of future in situ TEM are provided.« less

Perspectives on in situ electron microscopy

DOE PAGES

Zheng, Haimei; Zhu, Yimei

2017-03-29

In situ transmission electron microscopy (TEM) with the ability to reveal materials dynamic processes with high spatial and temporal resolution has attracted significant interest. The recent advances in in situ methods, including liquid and gas sample environment, pump-probe ultrafast microscopy, nanomechanics and ferroelectric domain switching the aberration corrected electron optics as well as fast electron detector has opened new opportunities to extend the impact of in situ TEM in broad areas of research ranging from materials science to chemistry, physics and biology. Here in this paper, we highlight the development of liquid environment electron microscopy and its applications in themore » study of colloidal nanoparticle growth, electrochemical processes and others; in situ study of topological vortices in ferroelectric and ferromagnetic materials. At the end, perspectives of future in situ TEM are provided.« less

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

A hard X-ray nanoprobe beamline for nanoscale microscopy

PubMed Central

Winarski, Robert P.; Holt, Martin V.; Rose, Volker; Fuesz, Peter; Carbaugh, Dean; Benson, Christa; Shu, Deming; Kline, David; Stephenson, G. Brian; McNulty, Ian; Maser, Jörg

2012-01-01

The Hard X-ray Nanoprobe Beamline (or Nanoprobe Beamline) is an X-ray microscopy facility incorporating diffraction, fluorescence and full-field imaging capabilities designed and operated by the Center for Nanoscale Materials and the Advanced Photon Source at Sector 26 of the Advanced Photon Source at Argonne National Laboratory. This facility was constructed to probe the nanoscale structure of biological, environmental and material sciences samples. The beamline provides intense focused X-rays to the Hard X-ray Nanoprobe (or Nanoprobe) which incorporates Fresnel zone plate optics and a precision laser sensing and control system. The beamline operates over X-ray energies from 3 to 30 keV, enabling studies of most elements in the periodic table, with a particular emphasis on imaging transition metals. PMID:23093770

Recent Advances in Nanodisc Technology for Membrane Proteins Studies (2012–2017)

PubMed Central

Rouck, John; Krapf, John; Roy, Jahnabi; Huff, Hannah; Das, Aditi

2017-01-01

Historically, progress in membrane protein research has been hindered due to solubility issues. The introduction of biomembrane mimetics has since stimulated the field’s momentum. One mimetic, the nanodisc, has proved to be an exceptional system for solubilizing membrane proteins. Herein, we critically evaluate the advantages and imperfections from employing nanodiscs in biophysical and biochemical studies. Specifically, we examine the techniques that have been modified to study membrane proteins in nanodiscs. Techniques discussed include fluorescence microscopy, solution state/solid state NMR, electron microscopy, SAXS, and several mass spectroscopy methods. Newer techniques such as SPR, charge sensitive optical detection, and scintillation proximity assays are also reviewed. Lastly, we cover nanodiscs advancing nanotechnology through nanoplasmonic biosensing, lipoprotein-nanoplatelets, and sortase-mediated labeling of nanodiscs. PMID:28581067

Recent advances in Lorentz microscopy

DOE PAGES

Phatak, C.; Petford-Long, A. K.; De Graef, M.

2016-01-05

Lorentz transmission electron microscopy (LTEM) has evolved from a qualitative magnetic domain observation technique to a quantitative technique for the determination of the magnetization state of a sample. Here, we describe recent developments in techniques and imaging modes, including the use of spherical aberration correction to improve the spatial resolution of LTEM into the single nanometer range, and novel in situ observation modes. We also review recent advances in the modeling of the wave optical magnetic phase shift as well as in the area of phase reconstruction by means of the Transport of Intensity Equation (TIE) approach, and discuss vectormore » field electron tomography, which has emerged as a powerful tool for the 3D reconstruction of magnetization configurations. Finally, we conclude this review with a brief overview of recent LTEM applications.« less

Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane.

PubMed

Stone, Matthew B; Shelby, Sarah A; Veatch, Sarah L

2017-06-14

Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.

Complex Light

NASA Astrophysics Data System (ADS)

Secor, Jeff; Alfano, Robert; Ashrafi, Solyman

2017-01-01

The emerging field of complex light-the study and application of custom light beams with tailored intensity, polarization or phase-is a focal point for fundamental breakthroughs in optical science. As this review will show, those advances in fundamental understanding, coupled with the latest developments in complex light generation, are translating into a range of diverse and cross-disciplinary applications that span microscopy, high-data-rate communications, optical trapping and quantum optics. We can expect more twists along the way, too, as researchers seek to manipulate and control the propagation speed of complex light beams, while others push the more exotic possibilities afforded by complex light in quantum-entanglement experiments.

Unconventional methods of imaging: computational microscopy and compact implementations

NASA Astrophysics Data System (ADS)

McLeod, Euan; Ozcan, Aydogan

2016-07-01

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

The use of FTIR microscopy for evaluation of herpes viruses infection development kinetics

NASA Astrophysics Data System (ADS)

Erukhimovitch, Vitaly; Mukmanov, Igor; Talyshinsky, Marina; Souprun, Yelena; Huleihel, Mahmoud

2004-08-01

The kinetics of Herpes simplex infection development was studied using an FTIR microscopy (FTIR-M) method. The family of herpes viruses includes several members like H. simplex types I and II (HSV I, II), Varicella zoster (VZV) viruses which are involved in various human and animal infections of different parts of the body. In our previous study, we found significant spectral differences between normal uninfected cells in cultures and cells infected with herpes viruses at early stages of the infection. In the present study, cells in cultures were infected with either HSV-I or VZV and at various times post-infection they were examined either by optical microscopy or by advanced FTIR-M. Spectroscopic measurements show a consistent decrease in the intensity of the carbohydrate peak in correlation with the viral infection development, observed by optical microscopy. This decrease in cellular carbohydrate level was used as indicator for herpes viruses infection kinetics. This parameter could be used as a basis for applying a spectroscopic method for the evaluation of herpes virus infection development. Our results show also that the development kinetics of viral infection has an exponential character for these viruses.

Advanced chemical imaging and comparison of human and porcine hair follicles for drug delivery by confocal Raman microscopy.

PubMed

Franzen, Lutz; Mathes, Christiane; Hansen, Steffi; Windbergs, Maike

2013-06-01

Hair follicles have recently gained a lot of interest for dermal drug delivery. They provide facilitated penetration into the skin and a high potential to serve as a drug depot. In this area of research, excised pig ear is a widely accepted in vitro model to evaluate penetration of drug delivery into hair follicles. However, a comparison of human and porcine follicles in terms of chemical composition has not been performed so far. In this study, we applied confocal Raman microscopy as a chemically selective imaging technique to compare human and porcine follicle composition and to visualize component distribution within follicle cross-sections. Based on the evaluation of human and porcine Raman spectra optical similarity for both species was successfully confirmed. Furthermore, cyanoacrylate skin surface biopsies, which are generally used to determine the extent of follicular penetration, were imaged by a novel complementary analytical approach combining confocal Raman microscopy and optical profilometry. This all-encompassing analysis allows investigation of intactness and component distribution of the excised hair bulb in three dimensions. Confocal Raman microscopy shows a high potential as a noninvasive and chemically selective technique for the analysis of trans-follicular drug delivery.

Advanced chemical imaging and comparison of human and porcine hair follicles for drug delivery by confocal Raman microscopy

NASA Astrophysics Data System (ADS)

Franzen, Lutz; Mathes, Christiane; Hansen, Steffi; Windbergs, Maike

2013-06-01

Hair follicles have recently gained a lot of interest for dermal drug delivery. They provide facilitated penetration into the skin and a high potential to serve as a drug depot. In this area of research, excised pig ear is a widely accepted in vitro model to evaluate penetration of drug delivery into hair follicles. However, a comparison of human and porcine follicles in terms of chemical composition has not been performed so far. In this study, we applied confocal Raman microscopy as a chemically selective imaging technique to compare human and porcine follicle composition and to visualize component distribution within follicle cross-sections. Based on the evaluation of human and porcine Raman spectra optical similarity for both species was successfully confirmed. Furthermore, cyanoacrylate skin surface biopsies, which are generally used to determine the extent of follicular penetration, were imaged by a novel complementary analytical approach combining confocal Raman microscopy and optical profilometry. This all-encompassing analysis allows investigation of intactness and component distribution of the excised hair bulb in three dimensions. Confocal Raman microscopy shows a high potential as a noninvasive and chemically selective technique for the analysis of trans-follicular drug delivery.

Digital micromirror devices: principles and applications in imaging.

PubMed

Bansal, Vivek; Saggau, Peter

2013-05-01

A digital micromirror device (DMD) is an array of individually switchable mirrors that can be used in many advanced optical systems as a rapid spatial light modulator. With a DMD, several implementations of confocal microscopy, hyperspectral imaging, and fluorescence lifetime imaging can be realized. The DMD can also be used as a real-time optical processor for applications such as the programmable array microscope and compressive sensing. Advantages and disadvantages of the DMD for these applications as well as methods to overcome some of the limitations will be discussed in this article. Practical considerations when designing with the DMD and sample optical layouts of a completely DMD-based imaging system and one in which acousto-optic deflectors (AODs) are used in the illumination pathway are also provided.

Nanoimprint of a 3D structure on an optical fiber for light wavefront manipulation.

PubMed

Calafiore, Giuseppe; Koshelev, Alexander; Allen, Frances I; Dhuey, Scott; Sassolini, Simone; Wong, Edward; Lum, Paul; Munechika, Keiko; Cabrini, Stefano

2016-09-16

Integration of complex photonic structures onto optical fiber facets enables powerful platforms with unprecedented optical functionalities. Conventional nanofabrication technologies, however, do not permit viable integration of complex photonic devices onto optical fibers owing to their low throughput and high cost. In this paper we report the fabrication of a three-dimensional structure achieved by direct nanoimprint lithography on the facet of an optical fiber. Nanoimprint processes and tools were specifically developed to enable a high lithographic accuracy and coaxial alignment of the optical device with respect to the fiber core. To demonstrate the capability of this new approach, a 3D beam splitter has been designed, imprinted and optically characterized. Scanning electron microscopy and optical measurements confirmed the good lithographic capabilities of the proposed approach as well as the desired optical performance of the imprinted structure. The inexpensive solution presented here should enable advancements in areas such as integrated optics and sensing, achieving enhanced portability and versatility of fiber optic components.

Attosecond electron pulses for 4D diffraction and microscopy

PubMed Central

Baum, Peter; Zewail, Ahmed H.

2007-01-01

In this contribution, we consider the advancement of ultrafast electron diffraction and microscopy to cover the attosecond time domain. The concept is centered on the compression of femtosecond electron packets to trains of 15-attosecond pulses by the use of the ponderomotive force in synthesized gratings of optical fields. Such attosecond electron pulses are significantly shorter than those achievable with extreme UV light sources near 25 nm (≈50 eV) and have the potential for applications in the visualization of ultrafast electron dynamics, especially of atomic structures, clusters of atoms, and some materials. PMID:18000040

Chemical mapping and quantification at the atomic scale by scanning transmission electron microscopy.

PubMed

Chu, Ming-Wen; Chen, Cheng Hsuan

2013-06-25

With innovative modern material-growth methods, a broad spectrum of fascinating materials with reduced dimensions-ranging from single-atom catalysts, nanoplasmonic and nanophotonic materials to two-dimensional heterostructural interfaces-is continually emerging and extending the new frontiers of materials research. A persistent central challenge in this grand scientific context has been the detailed characterization of the individual objects in these materials with the highest spatial resolution, a problem prompting the need for experimental techniques that integrate both microscopic and spectroscopic capabilities. To date, several representative microscopy-spectroscopy combinations have become available, such as scanning tunneling microscopy, tip-enhanced scanning optical microscopy, atom probe tomography, scanning transmission X-ray microscopy, and scanning transmission electron microscopy (STEM). Among these tools, STEM boasts unique chemical and electronic sensitivity at unparalleled resolution. In this Perspective, we elucidate the advances in STEM and chemical mapping applications at the atomic scale by energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy with a focus on the ultimate challenge of chemical quantification with atomic accuracy.

Boundary segmentation for fluorescence microscopy using steerable filters

NASA Astrophysics Data System (ADS)

Ho, David Joon; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.

2017-02-01

Fluorescence microscopy is used to image multiple subcellular structures in living cells which are not readily observed using conventional optical microscopy. Moreover, two-photon microscopy is widely used to image structures deeper in tissue. Recent advancement in fluorescence microscopy has enabled the generation of large data sets of images at different depths, times, and spectral channels. Thus, automatic object segmentation is necessary since manual segmentation would be inefficient and biased. However, automatic segmentation is still a challenging problem as regions of interest may not have well defined boundaries as well as non-uniform pixel intensities. This paper describes a method for segmenting tubular structures in fluorescence microscopy images of rat kidney and liver samples using adaptive histogram equalization, foreground/background segmentation, steerable filters to capture directional tendencies, and connected-component analysis. The results from several data sets demonstrate that our method can segment tubular boundaries successfully. Moreover, our method has better performance when compared to other popular image segmentation methods when using ground truth data obtained via manual segmentation.

Fibre-optic nonlinear optical microscopy and endoscopy.

PubMed

Fu, L; Gu, M

2007-06-01

Nonlinear optical microscopy has been an indispensable laboratory tool of high-resolution imaging in thick tissue and live animals. Rapid developments of fibre-optic components in terms of growing functionality and decreasing size provide enormous opportunities for innovations in nonlinear optical microscopy. Fibre-based nonlinear optical endoscopy is the sole instrumentation to permit the cellular imaging within hollow tissue tracts or solid organs that are inaccessible to a conventional optical microscope. This article reviews the current development of fibre-optic nonlinear optical microscopy and endoscopy, which includes crucial technologies for miniaturized nonlinear optical microscopy and their embodiments of endoscopic systems. A particular attention is given to several classes of photonic crystal fibres that have been applied to nonlinear optical microscopy due to their unique properties for ultrashort pulse delivery and signal collection. Furthermore, fibre-optic nonlinear optical imaging systems can be classified into portable microscopes suitable for imaging behaving animals, rigid endoscopes that allow for deep tissue imaging with minimally invasive manners, and flexible endoscopes enabling imaging of internal organs. Fibre-optic nonlinear optical endoscopy is coming of age and a paradigm shift leading to optical microscope tools for early cancer detection and minimally invasive surgery.

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.

A beginner's guide to atomic force microscopy probing for cell mechanics

PubMed Central

2016-01-01

Abstract Atomic Force microscopy (AFM) is becoming a prevalent tool in cell biology and biomedical studies, especially those focusing on the mechanical properties of cells and tissues. The newest generation of bio‐AFMs combine ease of use and seamless integration with live‐cell epifluorescence or more advanced optical microscopies. As a unique feature with respect to other bionanotools, AFM provides nanometer‐resolution maps for cell topography, stiffness, viscoelasticity, and adhesion, often overlaid with matching optical images of the probed cells. This review is intended for those about to embark in the use of bio‐AFMs, and aims to assist them in designing an experiment to measure the mechanical properties of adherent cells. In addition to describing the main steps in a typical cell mechanics protocol and explaining how data is analysed, this review will also discuss some of the relevant contact mechanics models available and how they have been used to characterize specific features of cellular and biological samples. Microsc. Res. Tech. 80:75–84, 2017. © 2016 Wiley Periodicals, Inc. PMID:27676584

Photoacoustics and speed-of-sound dual mode imaging with a long depth-of-field by using annular ultrasound array.

PubMed

Ding, Qiuning; Tao, Chao; Liu, Xiaojun

2017-03-20

Speed-of-sound and optical absorption reflect the structure and function of tissues from different aspects. A dual-mode microscopy system based on a concentric annular ultrasound array is proposed to simultaneously acquire the long depth-of-field images of speed-of-sound and optical absorption of inhomogeneous samples. First, speed-of-sound is decoded from the signal delay between each element of the annular array. The measured speed-of-sound could not only be used as an image contrast, but also improve the resolution and accuracy of spatial location of photoacoustic image in inhomogeneous acoustic media. Secondly, benefitting from dynamic focusing of annular array and the measured speed-of-sound, it is achieved an advanced acoustic-resolution photoacoustic microscopy with a precise position and a long depth-of-field. The performance of the dual-mode imaging system has been experimentally examined by using a custom-made annular array. The proposed dual-mode microscopy might have the significances in monitoring the biological physiological and pathological processes.

Application and Miniaturization of Linear and Nonlinear Raman Microscopy for Biomedical Imaging

NASA Astrophysics Data System (ADS)

Mittal, Richa

Current diagnostics for several disorders rely on surgical biopsy or evaluation of ex vivo bodily fluids, which have numerous drawbacks. We evaluated the potential for vibrational techniques (both linear and nonlinear Raman) as a reliable and noninvasive diagnostic tool. Raman spectroscopy is an optical technique for molecular analysis that has been used extensively in various biomedical applications. Based on demonstrated capabilities of Raman spectroscopy we evaluated the potential of the technique for providing a noninvasive diagnosis of mucopolysaccharidosis (MPS). These studies show that Raman spectroscopy can detect subtle changes in tissue biochemistry. In applications where sub-micrometer visualization of tissue compositional change is required, a transition from spectroscopy to high quality imaging is necessary. Nonlinear vibrational microscopy is sensitive to the same molecular vibrations as linear Raman, but features fast imaging capabilities. Coherent Raman scattering when combined with other nonlinear optical (NLO) techniques (like two-photon excited fluorescence and second harmonic generation) forms a collection of advanced optical techniques that provide noninvasive chemical contrast at submicron resolution. This capability to examine tissues without external molecular agents is driving the NLO approach towards clinical applications. However, the unique imaging capabilities of NLO microscopy are accompanied by complex instrument requirements. Clinical examination requires portable imaging systems for rapid inspection of tissues. Optical components utilized in NLO microscopy would then need substantial miniaturization and optimization to enable in vivo use. The challenges in designing compact microscope objective lenses and laser beam scanning mechanisms are discussed. The development of multimodal NLO probes for imaging oral cavity tissue is presented. Our prototype has been examined for ex vivo tissue imaging based on intrinsic fluorescence and SHG contrast. These studies show a potential for multiphoton compact probes to be used for real time imaging in the clinic.

Structural Investigation of Biological and Semiconductor Nanostructures with Nonlinear Multicontrast Microscopy

NASA Astrophysics Data System (ADS)

Cisek, Richard

Physical and functional properties of advanced nano-composite materials and biological structures are determined by self-organized atoms and molecules into nanostructures and in turn by microscopic organization of the nanostructures into assemblies of higher structural complexity. Therefore, microscopes are indispensable tools for structural investigations at various levels of organization. In this work, novel nonlinear optical microscopy methods were developed to non-invasively study structural organization at the nanoscopic and microscopic levels. Atomic organization of semiconductor nanowires, molecular organization of amylose biocrystallites in starch granules, and microscopic organization of several photosynthetic organisms was elucidated. The structure of ZnSe nanowires, key components in many modern nanodevices, was investigated using polarization harmonic generation microscopy. Based on nonlinear optical properties of the different crystal lattices, zinc blende and wurtzite nanowires were differentiated, and the three-dimensional orientation of the zinc blende nanowires could be found. The structure of starch granules, a model biocrystal, important in food as well as health sciences, was also investigated using polarization harmonic microscopy. The study was combined with ab initio calculations using the crystal structures of amylose A and B, revealing that second harmonic signals originate from the hydroxide and hydrogen bonds in the starch granules. Visualization of several photosynthetic organisms including the green algae, Chlamydomonas reinhardtii, two species of cyanobacteria, Leptolyngbya sp. and Anabaena sp., aggregates of light-harvesting pigment-protein complexes as well as chloroplasts from green plants were also explored, revealing that future nonlinear microscopy applications could include structural studies of cell walls, the Chlamydomonas eyespot, and photosynthetic membranes. In this study, several nonlinear optical microscopy modalities were developed for quantitative structural investigations of nano and micro-sized architectures. Non-invasive extraction of crystallographic information in microscopic samples will have a number of potential benefits, for example, in clinical applications, allowing observations of disease states inside tissues without the need for biopsy. Industrial nanotechnology will benefit from fast determination of nanostructures with nonlinear microscopy that will improve quality of nanodevices.

Applying tattoo dye as a third-harmonic generation contrast agent for in vivo optical virtual biopsy of human skin

NASA Astrophysics Data System (ADS)

Tsai, Ming-Rung; Lin, Chen-Yu; Liao, Yi-Hua; Sun, Chi-Kuang

2013-02-01

Third-harmonic generation (THG) microscopy has been reported to provide intrinsic contrast in elastic fibers, cytoplasmic membrane, nucleus, actin filaments, lipid bodies, hemoglobin, and melanin in human skin. For advanced molecular imaging, exogenous contrast agents are developed for a higher structural or molecular specificity. We demonstrate the potential of the commonly adopted tattoo dye as a THG contrast agent for in vivo optical biopsy of human skin. Spectroscopy and microscopy experiments were performed on cultured cells with tattoo dyes, in tattooed mouse skin, and in tattooed human skin to demonstrate the THG enhancement effect. Compared with other absorbing dyes or nanoparticles used as exogenous THG contrast agents, tattoo dyes are widely adopted in human skin so that future clinical biocompatibility evaluation is relatively achievable. Combined with the demonstrated THG enhancement effect, tattoo dyes show their promise for future clinical imaging applications.

Functional Scanning Probe Imaging of Nanostructured Solar Energy Materials.

PubMed

Giridharagopal, Rajiv; Cox, Phillip A; Ginger, David S

2016-09-20

From hybrid perovskites to semiconducting polymer/fullerene blends for organic photovoltaics, many new materials being explored for energy harvesting and storage exhibit performance characteristics that depend sensitively on their nanoscale morphology. At the same time, rapid advances in the capability and accessibility of scanning probe microscopy methods over the past decade have made it possible to study processing/structure/function relationships ranging from photocurrent collection to photocarrier lifetimes with resolutions on the scale of tens of nanometers or better. Importantly, such scanning probe methods offer the potential to combine measurements of local structure with local function, and they can be implemented to study materials in situ or devices in operando to better understand how materials evolve in time in response to an external stimulus or environmental perturbation. This Account highlights recent advances in the development and application of scanning probe microscopy methods that can help address such questions while filling key gaps between the capabilities of conventional electron microscopy and newer super-resolution optical methods. Focusing on semiconductor materials for solar energy applications, we highlight a range of electrical and optoelectronic scanning probe microscopy methods that exploit the local dynamics of an atomic force microscope tip to probe key properties of the solar cell material or device structure. We discuss how it is possible to extract relevant device properties using noncontact scanning probe methods as well as how these properties guide materials development. Specifically, we discuss intensity-modulated scanning Kelvin probe microscopy (IM-SKPM), time-resolved electrostatic force microscopy (trEFM), frequency-modulated electrostatic force microscopy (FM-EFM), and cantilever ringdown imaging. We explain these developments in the context of classic atomic force microscopy (AFM) methods that exploit the physics of cantilever motion and photocarrier generation to provide robust, nanoscale measurements of materials physics that are correlated with device operation. We predict that the multidimensional data sets made possible by these types of methods will become increasingly important as advances in data science expand capabilities and opportunities for image correlation and discovery.

Advances in thickness measurements and dynamic visualization of the tear film using non-invasive optical approaches.

PubMed

Bai, Yuqiang; Nichols, Jason J

2017-05-01

The thickness of tear film has been investigated under both invasive and non-invasive methods. While invasive methods are largely historical, more recent noninvasive methods are generally based on optical approaches that provide accurate, precise, and rapid measures. Optical microscopy, interferometry, and optical coherence tomography (OCT) have been developed to characterize the thickness of tear film or certain aspects of the tear film (e.g., the lipid layer). This review provides an in-depth overview on contemporary optical techniques used in studying the tear film, including both advantages and limitations of these approaches. It is anticipated that further developments of high-resolution OCT and other interferometric methods will enable a more accurate and precise measurement of the thickness of the tear film and its related dynamic properties. Copyright © 2017 Elsevier Ltd. All rights reserved.

Beyond sequencing: optical mapping of DNA in the age of nanotechnology and nanoscopy.

PubMed

Levy-Sakin, Michal; Ebenstein, Yuval

2013-08-01

Next generation sequencing (NGS) is revolutionizing all fields of biological research but it fails to extract the full range of information associated with genetic material. Optical mapping of DNA grants access to genetic and epigenetic information on individual DNA molecules up to ∼1 Mbp in length. Fluorescent labeling of specific sequence motifs, epigenetic marks and other genomic information on individual DNA molecules generates a high content optical barcode along the DNA. By stretching the DNA to a linear configuration this barcode may be directly visualized by fluorescence microscopy. We discuss the advances of these methods in light of recent developments in nano-fabrication and super-resolution optical imaging (nanoscopy) and review the latest achievements of optical mapping in the context of genomic analysis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

PubMed

Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

2017-06-28

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

Nonlinear Polarimetric Microscopy for Biomedical Imaging

NASA Astrophysics Data System (ADS)

Samim, Masood

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

Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening

PubMed Central

Galanzha, Ekaterina I; Tuchin, Valery V; Zharov, Vladimir P

2007-01-01

Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo. Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry, super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., real-time quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers, drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and high-speed imaging of cell transient deformability in flow. In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cell’s functional state (e.g., live, apoptotic, or necrotic), and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed. PMID:17226898

Dictionary of Microscopy

NASA Astrophysics Data System (ADS)

Heath, Julian

2005-10-01

The past decade has seen huge advances in the application of microscopy in all areas of science. This welcome development in microscopy has been paralleled by an expansion of the vocabulary of technical terms used in microscopy: terms have been coined for new instruments and techniques and, as microscopes reach even higher resolution, the use of terms that relate to the optical and physical principles underpinning microscopy is now commonplace. The Dictionary of Microscopy was compiled to meet this challenge and provides concise definitions of over 2,500 terms used in the fields of light microscopy, electron microscopy, scanning probe microscopy, x-ray microscopy and related techniques. Written by Dr Julian P. Heath, Editor of Microscopy and Analysis, the dictionary is intended to provide easy navigation through the microscopy terminology and to be a first point of reference for definitions of new and established terms. The Dictionary of Microscopy is an essential, accessible resource for: students who are new to the field and are learning about microscopes equipment purchasers who want an explanation of the terms used in manufacturers' literature scientists who are considering using a new microscopical technique experienced microscopists as an aide mémoire or quick source of reference librarians, the press and marketing personnel who require definitions for technical reports.

Lithographically-fabricated channel arrays for confocal x-ray fluorescence microscopy and XAFS

NASA Astrophysics Data System (ADS)

Woll, Arthur R.; Agyeman-Budu, David; Choudhury, Sanjukta; Coulthard, Ian; Finnefrock, Adam C.; Gordon, Robert; Hallin, Emil; Mass, Jennifer

2014-03-01

Confocal X-ray Fluorescence Microscopy (CXRF) employs overlapping focal regions of two x-ray optics—a condenser and collector—to directly probe a 3D volume. The minimum-achievable size of this probe volume is limited by the collector, for which polycapillaries are generally the optic of choice. Recently, we demonstrated an alternative collection optic for CXRF, consisting of an array of micron-scale collimating channels, etched in silicon, and arranged like spokes of a wheel directed towards a single source position. The optic, while successful, had a working distance of only 0.2 mm and exhibited relatively low total collection efficiency, limiting its practical application. Here, we describe a new design in which the collimating channels are formed by a staggered array of pillars whose side-walls taper away from the channel axis. This approach improves both collection efficiency and working distance, while maintaining excellent spatial resolution. We illustrate these improvements with confocal XRF data obtained at the Cornell High Energy Synchrotron Source (CHESS) and the Advanced Photon Source (APS) beamline 20-ID-B.

Advanced Imaging Approaches to Characterize Stromal and Metabolic Changes in In Vivo Mammary Tumor Models

DTIC Science & Technology

2015-02-01

Optical imaging , metabolism, tumor microenvironment, NADH, FAD, intravital imaging , collagen, metastasis 3.Overall Project Summary Our preliminary...Keely, KW Eliceiri. Novel Intravital Imaging Approaches to Characterize Collagen Alignment in Defined Mammary Tumor Models. Microscopy and...fixturing for intravital FLIM imaging through a rodent mammary imaging window. Stage is raised to accommodate tall 20xW objective. 14     Figure

Two-color CW STED nanoscopy

NASA Astrophysics Data System (ADS)

Chen, Xuanze; Liu, Yujia; Yang, Xusan; Wang, Tingting; Alonas, Eric; Santangelo, Philip J.; Ren, Qiushi; Xi, Peng

2013-02-01

Fluorescent microscopy has become an essential tool to study biological molecules, pathways and events in living cells, tissues and animals. Meanwhile even the most advanced confocal microscopy can only yield optical resolution approaching Abbe diffraction limit of 200 nm. This is still larger than many subcellular structures, which are too small to be resolved in detail. These limitations have driven the development of super-resolution optical imaging methodologies over the past decade. In stimulated emission depletion (STED) microscopy, the excitation focus is overlapped by an intense doughnut-shaped spot to instantly de-excite markers from their fluorescent state to the ground state by stimulated emission. This effectively eliminates the periphery of the Point Spread Function (PSF), resulting in a narrower focal region, or super-resolution. Scanning a sharpened spot through the specimen renders images with sub-diffraction resolution. Multi-color STED imaging can present important structural and functional information for protein-protein interaction. In this work, we presented a two-color, synchronization-free STED microscopy with a Ti:Sapphire oscillator. The excitation wavelengths were 532nm and 635nm, respectively. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. Human respiratory syncytial virus (hRSV) proteins were imaged with our two-color CW STED for co-localization analysis.

Three-dimensional image formation in fiber-optical second-harmonic-generation microscopy.

PubMed

Gu, Min; Fu, Ling

2006-02-06

Three-dimensional (3-D) image formation in fiber-optical second-harmonic-generation microscopy is revealed to be purely coherent and therefore can be described by a 3-D coherent transfer function (CTF) that exhibits the same spatial frequency passband as that of fiber-optical reflection-mode non-fluorescence microscopy. When the numerical aperture of the fiber is much larger than the angle of convergence of the illumination on the fiber aperture, the performance of fiber-optical second-harmonic-generation microscopy behaves as confocal second-harmonic-generation microscopy. The dependence of axial resolution on fiber coupling parameters shows an improvement of approximately 7%, compared with that in fiber-optical two-photon fluorescence microscopy.

Super-resolution atomic force photoactivated microscopy of biological samples (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lee, Seunghyun; Kim, Hyemin; Shin, Seungjun; Doh, Junsang; Kim, Chulhong

2017-03-01

Optical microscopy (OM) and photoacoustic microscopy (PAM) have previously been used to image the optical absorption of intercellular features of biological cells. However, the optical diffraction limit ( 200 nm) makes it difficult for these modalities to image nanoscale inner cell structures and the distribution of internal cell components. Although super-resolution fluorescence microscopy, such as stimulated emission depletion microscopy (STED) and stochastic optical reconstruction microscopy (STORM), has successfully performed nanoscale biological imaging, these modalities require the use of exogenous fluorescence agents, which are unfavorable for biological samples. Our newly developed atomic force photoactivated microscopy (AFPM) can provide optical absorption images with nanoscale lateral resolution without any exogenous contrast agents. AFPM combines conventional atomic force microscopy (AFM) and an optical excitation system, and simultaneously provides multiple contrasts, such as the topography and magnitude of optical absorption. AFPM can detect the intrinsic optical absorption of samples with 8 nm lateral resolution, easily overcoming the diffraction limit. Using the label-free AFPM system, we have successfully imaged the optical absorption properties of a single melanoma cell (B16F10) and a rosette leaf epidermal cell of Arabidopsis (ecotype Columbia (Col-0)) with nanoscale lateral resolution. The remarkable images show the melanosome distribution of a melanoma cell and the biological structures of a plant cell. AFPM provides superior imaging of optical absorption with a nanoscale lateral resolution, and it promises to become widely used in biological and chemical research.

Nanoscale nuclear architecture for cancer diagnosis by spatial-domain low-coherence quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Wang, Pin; Bista, Rajan K.; Khalbuss, Walid E.; Qiu, Wei; Staton, Kevin D.; Zhang, Lin; Brentnall, Teresa A.; Brand, Randall E.; Liu, Yang

2011-03-01

Alterations in nuclear architecture are the hallmark diagnostic characteristic of cancer cells. In this work, we show that the nuclear architectural characteristics quantified by spatial-domain low-coherence quantitative phase microscopy (SL-QPM), is more sensitive for the identification of cancer cells than conventional cytopathology. We demonstrated the importance of nuclear architectural characteristics in both an animal model of intestinal carcinogenesis - APC/Min mouse model and human cytology specimens with colorectal cancer by identifying cancer from cytologically noncancerous appearing cells. The determination of nanoscale nuclear architecture using this simple and practical optical instrument is a significant advance towards cancer diagnosis.

Corneal edema after phacoemulsification

PubMed Central

Sharma, Namrata; Singhal, Deepali; Nair, Sreelakshmi P; Sahay, Pranita; Sreeshankar, SS; Maharana, Prafulla Kumar

2017-01-01

Phacoemulsification is the most commonly performed cataract surgery in this era. With all the recent advances in investigations and management of cataract through phacoemulsification, most of the patients are able to achieve excellent visual outcome. Corneal edema after phacoemulsification in the immediate postoperative period often leads to patient dissatisfaction and worsening of outcome. Delayed onset corneal edema often warrants endothelial keratoplasty. This review highlights the etiopathogenesis, risk factors, and management of corneal edema in the acute phase including descemet's membrane detachment (DMD) and toxic anterior segment syndrome. Various investigative modalities such as pachymetry, specular microscopy, anterior segment optical coherence tomography, and confocal microscopy have been discussed briefly. PMID:29208818

Friction Stir Spot Welding of Advanced High Strength Steels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hovanski, Yuri; Grant, Glenn J.; Santella, M. L.

Friction stir spot welding techniques were developed to successfully join several advanced high strength steels. Two distinct tool materials were evaluated to determine the effect of tool materials on the process parameters and joint properties. Welds were characterized primarily via lap shear, microhardness, and optical microscopy. Friction stir spot welds were compared to the resistance spot welds in similar strength alloys by using the AWS standard for resistance spot welding high strength steels. As further comparison, a primitive cost comparison between the two joining processes was developed, which included an evaluation of the future cost prospects of friction stir spotmore » welding in advanced high strength steels.« less

The 2015 super-resolution microscopy roadmap

NASA Astrophysics Data System (ADS)

Hell, Stefan W.; Sahl, Steffen J.; Bates, Mark; Zhuang, Xiaowei; Heintzmann, Rainer; Booth, Martin J.; Bewersdorf, Joerg; Shtengel, Gleb; Hess, Harald; Tinnefeld, Philip; Honigmann, Alf; Jakobs, Stefan; Testa, Ilaria; Cognet, Laurent; Lounis, Brahim; Ewers, Helge; Davis, Simon J.; Eggeling, Christian; Klenerman, David; Willig, Katrin I.; Vicidomini, Giuseppe; Castello, Marco; Diaspro, Alberto; Cordes, Thorben

2015-11-01

Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio)physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of ‘super-resolution’ far-field optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough discussion on the concepts underlying super-resolution optical microscopy, the potential of different approaches, the importance of label optimization (such as reversible photoswitchable proteins) and applications in which these methods will have a significant impact. Mark Bates, Christian Eggeling

Dielectric Optical-Controllable Magnifying Lens by Nonlinear Negative Refraction

PubMed Central

Cao, Jianjun; Shang, Ce; Zheng, Yuanlin; Feng, Yaming; Chen, Xianfeng; Liang, Xiaogan; Wan, Wenjie

2015-01-01

A simple optical lens plays an important role for exploring the microscopic world in science and technology by refracting light with tailored spatially varying refractive indices. Recent advancements in nanotechnology enable novel lenses, such as, superlens and hyperlens, with sub-wavelength resolution capabilities by specially designed materials’ refractive indices with meta-materials and transformation optics. However, these artificially nano- or micro-engineered lenses usually suffer high losses from metals and are highly demanding in fabrication. Here, we experimentally demonstrate, for the first time, a nonlinear dielectric magnifying lens using negative refraction by degenerate four-wave mixing in a plano-concave glass slide, obtaining magnified images. Moreover, we transform a nonlinear flat lens into a magnifying lens by introducing transformation optics into the nonlinear regime, achieving an all-optical controllable lensing effect through nonlinear wave mixing, which may have many potential applications in microscopy and imaging science. PMID:26149952

Enhancing nanoparticle electrodynamics with gold nanoplate mirrors.

PubMed

Yan, Zijie; Bao, Ying; Manna, Uttam; Shah, Raman A; Scherer, Norbert F

2014-05-14

Mirrors and optical cavities can modify and enhance matter-radiation interactions. Here we report that chemically synthesized Au nanoplates can serve as micrometer-size mirrors that enhance electrodynamic interactions. Because of their plasmonic properties, the Au nanoplates enhance the brightness of scattered light from Ag nanoparticles near the nanoplate surface in dark-field microscopy. More importantly, enhanced optical trapping and optical binding of Ag nanoparticles are demonstrated in interferometric optical traps created from a single laser beam and its reflection from individual Au nanoplates. The enhancement of the interparticle force constant is ≈20-fold more than expected from the increased intensity due to standing wave interference. We show that the additional stability for optical binding arises from the restricted axial thermal motion of the nanoparticles that couples to and reduces the fluctuations in the lateral plane. This new mechanism greatly advances the photonic synthesis of ultrastable nanoparticle arrays and investigation of their properties.

Microscopy techniques in flavivirus research.

PubMed

Chong, Mun Keat; Chua, Anthony Jin Shun; Tan, Terence Tze Tong; Tan, Suat Hoon; Ng, Mah Lee

2014-04-01

The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus-host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses. Copyright © 2013 Elsevier Ltd. All rights reserved.

A commercialized photoacoustic microscopy system with switchable optical and acoustic resolutions

NASA Astrophysics Data System (ADS)

Pu, Yang; Bi, Renzhe; Olivo, Malini; Zhao, Xiaojie

2018-02-01

A focused-scanning photoacoustic microscopy (PAM) is available to help advancing life science research in neuroscience, cell biology, and in vivo imaging. At this early stage, the only one manufacturer of PAM systems, MicroPhotoAcoustics (MPA; Ronkonkoma, NY), MPA has developed a commercial PAM system with switchable optical and acoustic resolution (OR- and AR-PAM), using multiple patents licensed from the lab of Lihong Wang, who pioneered photoacoustics. The system includes different excitation sources. Two kilohertz-tunable, Q-switched, Diode Pumped Solid-State (DPSS) lasers offering a up to 30kHz pulse repetition rate and 9 ns pulse duration with 532 and 559 nm to achieve functional photoacoustic tomography for sO2 (oxygen saturation of hemoglobin) imaging in OR-PAM. A Ti:sapphire laser from 700 to 900 nm to achieve deep-tissue imaging. OR-PAM provides up to 1 mm penetration depth and 5 μm lateral resolution. while AR-PAM offers up to 3 mm imaging depth and 45 μm lateral resolution. The scanning step sizes for OR- and AR-PAM are 0.625 and 6.25 μm, respectively. Researchers have used the system for a range of applications, including preclinical neural imaging; imaging of cell nuclei in intestine, ear, and leg; and preclinical human imaging of finger cuticle. With the continuation of new technological advancements and discoveries, MPA plans to further advance PAM to achieve faster imaging speed, higher spatial resolution at deeper tissue layer, and address a broader range of biomedical applications.

DotLens smartphone microscopy for biological and biomedical applications (Conference Presentation)

NASA Astrophysics Data System (ADS)

Sung, Yu-Lung; Zhao, Fusheng; Shih, Wei-Chuan

2017-02-01

Recent advances in inkjet-printed optics have created a new class of lens fabrication technique. Lenses with a tunable geometry, magnification, and focal length can be fabricated by dispensing controlled amounts of liquid polymer onto a heated surface. This fabrication technique is highly cost-effective, and can achieve optically smooth surface finish. Dubbed DotLens, a single of which weighs less than 50 mg and occupies a volume less than 50 μL. DotLens can be attached onto any smartphone camera akin to a contact lens, and enable smartphones to obtain image resolution as fine as 1 µm. The surface curvature modifies the optical path of light to the image sensor, and enables the camera to focus as close as 2 mm. This enables microscopic imaging on a smartphone without any additional attachments, and has shown great potential in mobile point-of-care diagnostic systems, particularly for histology of tissue sections and cytology of blood cells. DotLens Smartphone Microscopy represents an innovative approach fundamentally different from other smartphone microscopes. In this paper, we describe the application and performance of DotLens smartphone microscopy in biological and biomedical research. In particular, we show recent results from images collected from pathology tissue slides with cancer features. In addition, we show performance in cytological analysis of blood smear. This tool has empowered Citizen Science investigators to collect microscopic images from various interesting objects.

Real-time visualization of melanin granules in normal human skin using combined multiphoton and reflectance confocal microscopy.

PubMed

Majdzadeh, Ali; Lee, Anthony M D; Wang, Hequn; Lui, Harvey; McLean, David I; Crawford, Richard I; Zloty, David; Zeng, Haishan

2015-05-01

Recent advances in biomedical optics have enabled dermal and epidermal components to be visualized at subcellular resolution and assessed noninvasively. Multiphoton microscopy (MPM) and reflectance confocal microscopy (RCM) are noninvasive imaging modalities that have demonstrated promising results in imaging skin micromorphology, and which provide complementary information regarding skin components. This study assesses whether combined MPM/RCM can visualize intracellular and extracellular melanin granules in the epidermis and dermis of normal human skin. We perform MPM and RCM imaging of in vivo and ex vivo skin in the infrared domain. The inherent three-dimensional optical sectioning capability of MPM/RCM is used to image high-contrast granular features across skin depths ranging from 50 to 90 μm. The optical images thus obtained were correlated with conventional histologic examination including melanin-specific staining of ex vivo specimens. MPM revealed highly fluorescent granular structures below the dermal-epidermal junction (DEJ) region. Histochemical staining also demonstrated melanin-containing granules that correlate well in size and location with the granular fluorescent structures observed in MPM. Furthermore, the MPM fluorescence excitation wavelength and RCM reflectance of cell culture-derived melanin were equivalent to those of the granules. This study suggests that MPM can noninvasively visualize and quantify subepidermal melanin in situ. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Nonlinear Optical Properties of Carotenoid and Chlorophyll Harmonophores

NASA Astrophysics Data System (ADS)

Tokarz, Danielle Barbara

Information regarding the structure and function of living tissues and cells is instrumental to the advancement of cell biology and biophysics. Nonlinear optical microscopy can provide such information, but only certain biological structures generate nonlinear optical signals. Therefore, structural specificity can be achieved by introducing labels for nonlinear optical microscopy. Few studies exist in the literature about labels that facilitate harmonic generation, coined "harmonophores". This thesis consists of the first major investigation of harmonophores for third harmonic generation (THG) microscopy. Carotenoids and chlorophylls were investigated as potential harmonophores. Their nonlinear optical properties were studied by the THG ratio technique. In addition, a tunable refractometer was built in order to determine their second hyperpolarizability (gamma). At 830 nm excitation wavelength, carotenoids and chlorophylls were found to have large negative gamma values however, at 1028 nm, the sign of gamma reversed for carotenoids and remained negative for chlorophylls. Consequently, at 1028 nm wavelength, THG signal is canceled with mixtures of carotenoids and chlorophylls. Furthermore, when such molecules are covalently bonded as dyads or interact within photosynthetic pigment-protein complexes, it is found that additive effects with the gamma values still play a role, however, the overall gamma value is also influenced by the intra-pigment and inter-pigment interaction. The nonlinear optical properties of aggregates containing chlorophylls and carotenoids were the target of subsequent investigations. Carotenoid aggregates were imaged with polarization-dependent second harmonic generation and THG microscopy. Both techniques revealed crystallographic information pertaining to H and J aggregates and beta-carotene crystalline aggregates found in orange carrot. In order to demonstrate THG enhancement due to labeling, cultured cells were labeled with carotenoid incorporated liposomes. In addition, Drosophila melanogaster larvae muscle as well as keratin structures in the hair cortex were labeled with beta-carotene. Polarization-dependent THG studies may be particularly useful in understanding the structural organization that occurs within biological structures containing carotenoids and chlorophylls such as photosynthetic pigment-protein complexes and carotenoid aggregates in plants and alga. Further, artificial labeling with carotenoids and chlorophylls may be useful in clinical applications since they are nontoxic, nutritionally valuable, and they can aid in visualizing structural changes in cellular components.

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

PubMed

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

2014-01-01

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

Nanostitched Composites with Improved Interlaminar and Intralaminar Strengths for Advanced Airframes in Sea-based Aviation

DTIC Science & Technology

2017-04-13

experimental  and  engineering  expertise...ex   situ,  and   preliminary  in   situ,   experimental   program   utilizing  optical  microscopy,   scanning  electron...modeling,  as  well  as   through  working  with  NAVAIR   for  guidance  on  several   topics  including   experimental

In vivo imaging of neural activity

PubMed Central

Yang, Weijian; Yuste, Rafael

2017-01-01

Since the introduction of calcium imaging to monitor neuronal activity with single-cell resolution, optical imaging methods have revolutionized neuroscience by enabling systematic recordings of neuronal circuits in living animals. The plethora of methods for functional neural imaging can be daunting to the nonexpert to navigate. Here we review advanced microscopy techniques for in vivo functional imaging and offer guidelines for which technologies are best suited for particular applications. PMID:28362436

Near- and far-field spectroscopic imaging investigation of resonant square-loop infrared metasurfaces.

PubMed

D' Archangel, Jeffrey; Tucker, Eric; Kinzel, Ed; Muller, Eric A; Bechtel, Hans A; Martin, Michael C; Raschke, Markus B; Boreman, Glenn

2013-07-15

Optical metamaterials have unique properties which result from geometric confinement of the optical conductivity. We developed a series of infrared metasurfaces based on an array of metallic square loop antennas. The far-field absorption spectrum can be designed with resonances across the infrared by scaling the geometric dimensions. We measure the amplitude and phase of the resonant mode as standing wave patterns within the square loops using scattering-scanning near-field optical microscopy (s-SNOM). Further, using a broad-band synchrotron-based FTIR microscope and s-SNOM at the Advanced Light Source, we are able to correlate far-field spectra to near-field modes of the metasurface as the resonance is tuned between samples. The results highlight the importance of multi-modal imaging for the design and characterization of optical metamaterials.

Large optical nonlinearity of indium tin oxide in its epsilon-near-zero region.

PubMed

Alam, M Zahirul; De Leon, Israel; Boyd, Robert W

2016-05-13

Nonlinear optical phenomena are crucial for a broad range of applications, such as microscopy, all-optical data processing, and quantum information. However, materials usually exhibit a weak optical nonlinearity even under intense coherent illumination. We report that indium tin oxide can acquire an ultrafast and large intensity-dependent refractive index in the region of the spectrum where the real part of its permittivity vanishes. We observe a change in the real part of the refractive index of 0.72 ± 0.025, corresponding to 170% of the linear refractive index. This change in refractive index is reversible with a recovery time of about 360 femtoseconds. Our results offer the possibility of designing material structures with large ultrafast nonlinearity for applications in nanophotonics. Copyright © 2016, American Association for the Advancement of Science.

Microstructural Evolution and Fracture Behavior of Friction-Stir-Welded Al-Cu Laminated Composites

NASA Astrophysics Data System (ADS)

Beygi, R.; Kazeminezhad, Mohsen; Kokabi, A. H.

2014-01-01

In this study, we attempt to characterize the microstructural evolution during friction stir butt welding of Al-Cu-laminated composites and its effect on the fracture behavior of the joint. Emphasis is on the material flow and particle distribution in the stir zone. For this purpose, optical microscopy and scanning electron microscopy (SEM) images, energy-dispersive spectroscopy EDS and XRD analyses, hardness measurements, and tensile tests are carried out on the joints. It is shown that intermetallic compounds exist in lamellas of banding structure formed in the advancing side of the welds. In samples welded from the Cu side, the banding structure in the advancing side and the hook formation in the retreating side determine the fracture behavior of the joint. In samples welded from the Al side, a defect is formed in the advancing side of the weld, which is attributed to insufficient material flow. It is concluded that the contact surface of the laminate (Al or Cu) with the shoulder of the FSW tool influences the material flow and microstructure of welds.

Biophotonics for imaging and cell manipulation: quo vadis?

NASA Astrophysics Data System (ADS)

Serafetinides, Alexandros A.; Makropoulou, Mirsini; Kotsifaki, Domna G.; Tsigaridas, Giorgos

2016-01-01

As one of the major health problems for mankind is cancer, any development for the early detection and effective treatment of cancer is crucial to saving lives. Worldwide, the dream for the anti-cancer procedure of attack is the development of a safe and efficient early diagnosis technique, the so called "optical biopsy". As early diagnosis of cancer is associated with improved prognosis, several laser based optical diagnostic methods were developed to enable earlier, non-invasive detection of human cancer, as Laser Induced Fluorescence spectroscopy (LIFs), Diffuse Reflectance spectroscopy (DRs), confocal microscopy, and Optical Coherence Tomography (OCT). Among them, Optical Coherence Tomography (OCT) imaging is considered to be a useful tool to differentiate healthy from malignant (e.g. basal cell carcinoma, squamous cell carcinoma) skin tissue. If the demand is to perform imaging in sub-tissular or even sub-cellular level, optical tweezers and atomic force microscopy have enabled the visualization of molecular events underlying cellular processes in live cells, as well as the manipulation and characterization of microscale or even nanoscale biostructures. In this work, we will present the latest advances in the field of laser imaging and manipulation techniques, discussing some representative experimental data focusing on the 21th century biophotonics roadmap of novel diagnostic and therapeutical approaches. As an example of a recently discussed health and environmental problem, we studied both experimentally and theoretically the optical trapping forces exerted on yeast cells and modified with estrogen-like acting compounds yeast cells, suspended in various buffer media.

Nonlinear optical microscopy for immunoimaging: a custom optimized system of high-speed, large-area, multicolor imaging

PubMed Central

Li, Hui; Cui, Quan; Zhang, Zhihong; Luo, Qingming

2015-01-01

Background The nonlinear optical microscopy has become the current state-of-the-art for intravital imaging. Due to its advantages of high resolution, superior tissue penetration, lower photodamage and photobleaching, as well as intrinsic z-sectioning ability, this technology has been widely applied in immunoimaging for a decade. However, in terms of monitoring immune events in native physiological environment, the conventional nonlinear optical microscope system has to be optimized for live animal imaging. Generally speaking, three crucial capabilities are desired, including high-speed, large-area and multicolor imaging. Among numerous high-speed scanning mechanisms used in nonlinear optical imaging, polygon scanning is not only linearly but also dispersion-freely with high stability and tunable rotation speed, which can overcome disadvantages of multifocal scanning, resonant scanner and acousto-optical deflector (AOD). However, low frame rate, lacking large-area or multicolor imaging ability make current polygonbased nonlinear optical microscopes unable to meet the requirements of immune event monitoring. Methods We built up a polygon-based nonlinear optical microscope system which was custom optimized for immunoimaging with high-speed, large-are and multicolor imaging abilities. Results Firstly, we validated the imaging performance of the system by standard methods. Then, to demonstrate the ability to monitor immune events, migration of immunocytes observed by the system based on typical immunological models such as lymph node, footpad and dorsal skinfold chamber are shown. Finally, we take an outlook for the possible advance of related technologies such as sample stabilization and optical clearing for more stable and deeper intravital immunoimaging. Conclusions This study will be helpful for optimizing nonlinear optical microscope to obtain more comprehensive and accurate information of immune events. PMID:25694951

Analysis of live cell images: Methods, tools and opportunities.

PubMed

Nketia, Thomas A; Sailem, Heba; Rohde, Gustavo; Machiraju, Raghu; Rittscher, Jens

2017-02-15

Advances in optical microscopy, biosensors and cell culturing technologies have transformed live cell imaging. Thanks to these advances live cell imaging plays an increasingly important role in basic biology research as well as at all stages of drug development. Image analysis methods are needed to extract quantitative information from these vast and complex data sets. The aim of this review is to provide an overview of available image analysis methods for live cell imaging, in particular required preprocessing image segmentation, cell tracking and data visualisation methods. The potential opportunities recent advances in machine learning, especially deep learning, and computer vision provide are being discussed. This review includes overview of the different available software packages and toolkits. Copyright © 2017. Published by Elsevier Inc.

Topological study of nanomaterials using surface-enhanced ellipsometric contrast microscopy (SEEC)

NASA Astrophysics Data System (ADS)

Muckenhirn, Sylvain

2016-03-01

Innovations in nanotechnology are empowering scientists to deepen their understanding of physical, chemical and biological mechanisms. Powerful and precise characterization systems are essential to meet researchers' requirements. SEEC (Surface Enhanced Ellipsometric Contrast) microscopy is an innovative advanced optical technique based on ellipsometric and interference fringes of Fizeau principles. This technique offers live and label-free topographic imaging of organic, inorganic and biological samples with high Z resolution (down to 0.1nm thickness), and enhanced X-Y detection limit (down to 1.5nm width). This technique has been successfully applied to the study of nanometric films and structures, biological layers, and nano-objects. We applied SEEC technology to different applications explored below.

Seeing cilia: imaging modalities for ciliary motion and clinical connections.

PubMed

Peabody, Jacelyn E; Shei, Ren-Jay; Bermingham, Brent M; Phillips, Scott E; Turner, Brett; Rowe, Steven M; Solomon, George M

2018-06-01

The respiratory tract is lined with multiciliated epithelial cells that function to move mucus and trapped particles via the mucociliary transport apparatus. Genetic and acquired ciliopathies result in diminished mucociliary clearance, contributing to disease pathogenesis. Recent innovations in imaging technology have advanced our understanding of ciliary motion in health and disease states. Application of imaging modalities including transmission electron microscopy, high-speed video microscopy, and micron-optical coherence tomography could improve diagnostics and be applied for precision medicine. In this review, we provide an overview of ciliary motion, imaging modalities, and ciliopathic diseases of the respiratory system including primary ciliary dyskinesia, cystic fibrosis, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis.

Functional Scanning Probe Imaging of Nanostructured Solar Energy Materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giridharagopal, Rajiv; Cox, Phillip A.; Ginger, David S.

From hybrid perovskites to semiconducting polymer/fullerene blends for organic photovoltaics, many new materials being explored for energy harvesting and storage exhibit performance characteristics that depend sensitively on their nanoscale morphology. At the same time, rapid advances in the capability and accessibility of scanning probe microscopy methods over the past decade have made it possible to study processing/structure/function relationships ranging from photocurrent collection to photocarrier lifetimes with resolutions on the scale of tens of nanometers or better. Importantly, such scanning probe methods offer the potential to combine measurements of local structure with local function, and they can be implemented to studymore » materials in situ or devices in operando to better understand how materials evolve in time in response to an external stimulus or environmental perturbation. This Account highlights recent advances in the development and application of scanning probe microscopy methods that can help address such questions while filling key gaps between the capabilities of conventional electron microscopy and newer super-resolution optical methods. Focusing on semiconductor materials for solar energy applications, we highlight a range of electrical and optoelectronic scanning probe microscopy methods that exploit the local dynamics of an atomic force microscope tip to probe key properties of the solar cell material or device structure. We discuss how it is possible to extract relevant device properties using noncontact scanning probe methods as well as how these properties guide materials development. Specifically, we discuss intensity-modulated scanning Kelvin probe microscopy (IM-SKPM), time-resolved electrostatic force microscopy (trEFM), frequency-modulated electrostatic force microscopy (FM-EFM), and cantilever ringdown imaging. We explain these developments in the context of classic atomic force microscopy (AFM) methods that exploit the physics of cantilever motion and photocarrier generation to provide robust, nanoscale measurements of materials physics that are correlated with device operation. We predict that the multidimensional data sets made possible by these types of methods will become increasingly important as advances in data science expand capabilities and opportunities for image correlation and discovery.« less

Functional Scanning Probe Imaging of Nanostructured Solar Energy Materials

DOE PAGES

Giridharagopal, Rajiv; Cox, Phillip A.; Ginger, David S.

2016-08-30

From hybrid perovskites to semiconducting polymer/fullerene blends for organic photovoltaics, many new materials being explored for energy harvesting and storage exhibit performance characteristics that depend sensitively on their nanoscale morphology. At the same time, rapid advances in the capability and accessibility of scanning probe microscopy methods over the past decade have made it possible to study processing/structure/function relationships ranging from photocurrent collection to photocarrier lifetimes with resolutions on the scale of tens of nanometers or better. Importantly, such scanning probe methods offer the potential to combine measurements of local structure with local function, and they can be implemented to studymore » materials in situ or devices in operando to better understand how materials evolve in time in response to an external stimulus or environmental perturbation. This Account highlights recent advances in the development and application of scanning probe microscopy methods that can help address such questions while filling key gaps between the capabilities of conventional electron microscopy and newer super-resolution optical methods. Focusing on semiconductor materials for solar energy applications, we highlight a range of electrical and optoelectronic scanning probe microscopy methods that exploit the local dynamics of an atomic force microscope tip to probe key properties of the solar cell material or device structure. We discuss how it is possible to extract relevant device properties using noncontact scanning probe methods as well as how these properties guide materials development. Specifically, we discuss intensity-modulated scanning Kelvin probe microscopy (IM-SKPM), time-resolved electrostatic force microscopy (trEFM), frequency-modulated electrostatic force microscopy (FM-EFM), and cantilever ringdown imaging. We explain these developments in the context of classic atomic force microscopy (AFM) methods that exploit the physics of cantilever motion and photocarrier generation to provide robust, nanoscale measurements of materials physics that are correlated with device operation. We predict that the multidimensional data sets made possible by these types of methods will become increasingly important as advances in data science expand capabilities and opportunities for image correlation and discovery.« less

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

Submicron-resolution photoacoustic microscopy of endogenous light-absorbing biomolecules

NASA Astrophysics Data System (ADS)

Zhang, Chi

Photoacoustic imaging in biomedicine has the unique advantage of probing endogenous light absorbers at various length scales with a 100% relative sensitivity. Among the several modalities of photoacoustic imaging, optical-resolution photoacoustic microscopy (OR-PAM) can achieve high spatial resolution, on the order of optical wavelength, at <1 mm depth in biological tissue (the optical ballistic regime). OR-PAM has been applied successfully to structural and functional imaging of blood vasculature and red blood cells in vivo. Any molecules which absorb sufficient light at certain wavelengths can potentially be imaged by PAM. Compared with pure optical imaging, which typically targets fluorescent markers, label-free PAM avoids the major concerns that the fluorescent labeling probes may disturb the function of biomolecules and may have an insufficient density. This dissertation aims to advance label-free OR-PAM to the subcellular scale. The first part of this dissertation describes the technological advancement of PAM yielding high spatial resolution in 3D. The lateral resolution was improved by using optical objectives with high numerical apertures for optical focusing. The axial resolution was improved by using broadband ultrasonic transducers for ultrasound detection. We achieved 220 nm lateral resolution in transmission mode, 0.43 microm lateral resolution in reflection mode, 7.6 microm axial resolution in normal tissue, and 5.8 microm axial resolution with silicone oil immersion/injection. The achieved lateral resolution and axial resolution were the finest reported at the time. With high-resolution in 3D, PAM was demonstrated to resolve cellular and subcellular structures in vivo, such as red blood cells and melanosomes in melanoma cells. Compared with previous PAM systems, our high-resolution PAM could resolve capillaries in mouse ears more clearly. As an example application, we demonstrated intracellular temperature imaging, assisted by fluorescence signal detection, with sub-degree temperature resolution and sub-micron lateral resolution. The second part of this dissertation describes the exploration of endogenous light-absorbing biomolecules for PAM. We demonstrated cytochromes and myoglobin as new absorption contrasts for PAM and identified the corresponding optimal wavelengths for imaging. Fixed fibroblasts on slides and mouse ear sections were imaged by PAM at 422 nm and 250 nm wavelengths to reveal cytoplasms and nuclei, respectively, as confirmed by standard hematoxylin and eosin (H&E) histology. By imaging a blood-perfused mouse heart at 532 nm down to 150 microm in depth, we derived the myocardial sheet thickness and the cleavage height from an undehydrated heart for the first time. The findings promote PAM at new wavelengths and open up new possibilities for characterizing biological tissue. Of particular interest, dual-wavelength PAM around 250 nm and 420 nm wavelengths is analogous to H&E histology. The last part of this dissertation describes the development of sectioning photoacoustic microscopy (SPAM), based on the advancement in spatial resolution and new contrasts for PAM, with applications in brain histology. Label-free SPAM, assisted by a microtome, acquires serial distortion-free images of a specimen on the surface. By exciting cell nuclei at 266 nm wavelength with high resolution, SPAM could pinpoint cell nuclei sensitively and specifically in the mouse brain section, as confirmed by H&E histology. SPAM was demonstrated to generate high-resolution 3D images, highlighting cell nuclei, of formalin-fixed paraffin-embedded mouse brains without tissue staining or clearing. SPAM can potentially serve as a high-throughput and minimal-artifact substitute for histology, probe many other biomolecules and cells, and become a universal tool for animal or human whole-organ microscopy, with diverse applications in life sciences.

Gain-guided soliton fiber laser with high-quality rectangle spectrum for ultrafast time-stretch microscopy.

PubMed

Hu, Song; Yao, Jian; Liu, Meng; Luo, Ai-Ping; Luo, Zhi-Chao; Xu, Wen-Cheng

2016-05-16

The ultrafast time-stretch microscopy has been proposed to enhance the temporal resolution of a microscopy system. The optical source is a key component for ultrafast time-stretch microscopy system. Herein, we reported on the gain-guided soliton fiber laser with high-quality rectangle spectrum for ultrafast time-stretch microscopy. By virtue of the excellent characteristics of the gain-guided soliton, the output power and the 3-dB bandwidth of the stable mode-locked soliton could be up to 3 mW and 33.7 nm with a high-quality rectangle shape, respectively. With the proposed robust optical source, the ultrafast time-stretch microscopy with the 49.6 μm resolution and a scan rate of 11 MHz was achieved without the external optical amplification. The obtained results demonstrated that the gain-guided soliton fiber laser could be used as an alternative high-quality optical source for ultrafast time-stretch microscopy and will introduce some applications in fields such as biology, chemical, and optical sensing.

Multi-MHz laser-scanning single-cell fluorescence microscopy by spatiotemporally encoded virtual source array

PubMed Central

Wu, Jianglai; Tang, Anson H. L.; Mok, Aaron T. Y.; Yan, Wenwei; Chan, Godfrey C. F.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2017-01-01

Apart from the spatial resolution enhancement, scaling of temporal resolution, equivalently the imaging throughput, of fluorescence microscopy is of equal importance in advancing cell biology and clinical diagnostics. Yet, this attribute has mostly been overlooked because of the inherent speed limitation of existing imaging strategies. To address the challenge, we employ an all-optical laser-scanning mechanism, enabled by an array of reconfigurable spatiotemporally-encoded virtual sources, to demonstrate ultrafast fluorescence microscopy at line-scan rate as high as 8 MHz. We show that this technique enables high-throughput single-cell microfluidic fluorescence imaging at 75,000 cells/second and high-speed cellular 2D dynamical imaging at 3,000 frames per second, outperforming the state-of-the-art high-speed cameras and the gold-standard laser scanning strategies. Together with its wide compatibility to the existing imaging modalities, this technology could empower new forms of high-throughput and high-speed biological fluorescence microscopy that was once challenged. PMID:28966855

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

NASA Astrophysics Data System (ADS)

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

2016-03-01

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

High-Resolution Methods for Diagnosing Cartilage Damage In Vivo

PubMed Central

Novakofski, Kira D.; Pownder, Sarah L.; Koff, Matthew F.; Williams, Rebecca M.; Potter, Hollis G.; Fortier, Lisa A.

2016-01-01

Advances in current clinical modalities, including magnetic resonance imaging and computed tomography, allow for earlier diagnoses of cartilage damage that could mitigate progression to osteoarthritis. However, current imaging modalities do not detect submicrometer damage. Developments in in vivo or arthroscopic techniques, including optical coherence tomography, ultrasonography, bioelectricity including streaming potential measurement, noninvasive electroarthrography, and multiphoton microscopy can detect damage at an earlier time point, but they are limited by a lack of penetration and the ability to assess an entire joint. This article reviews current advancements in clinical and developing modalities that can aid in the early diagnosis of cartilage injury and facilitate studies of interventional therapeutics. PMID:26958316

Use of Complementary Approaches to Imaging Biomolecules and Endogenous and Exogenous Trace Elements and Nanoparticles in Biological Samples

NASA Astrophysics Data System (ADS)

Brown, Koshonna Dinettia

X-ray Fluorescence Microscopy (XFM) is a useful technique for study of biological samples. XFM was used to map and quantify endogenous biological elements as well as exogenous materials in biological samples, such as the distribution of titanium dioxide (TiO2) nanoparticles. TiO 2 nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic particles for cancer detection and treatment, drug delivery, and induction of DNA breaks. Delivery of such nanoparticles can be targeted to specific cells and subcellular structures. In this work, we develop two novel approaches to stain TiO2 nanoparticles for optical microscopy and to confirm that staining by XFM. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called CLICK chemistry, for labeling of azide conjugated TiO2 nanoparticles with "clickable" dyes such as alkyne Alexa Fluor dyes with a high fluorescent yield. To confirm that the optical fluorescence signals of nanoparticles stained in situ match the distribution of the Ti element, we used high resolution synchrotron X-Ray Fluorescence Microscopy (XFM) using the Bionanoprobe instrument at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific X-ray fluorescence showed excellent overlap with the location of Alexa Fluor optical fluorescence detected by confocal microscopy. In this work XFM was also used to investigate native elemental differences between two different types of head and neck cancer, one associated with human papilloma virus infection, the other virus free. Future work may see a cross between these themes, for example, exploration of TiO2 nanoparticles as anticancer treatment for these two different types of head and neck cancer.

Shedding new light on lipid functions with CARS and SRS microscopy

PubMed Central

Yu, Yong; Ramachandran, Prasanna V.; Wang, Meng C.

2014-01-01

Modern optical microscopy has granted biomedical scientists unprecedented access to the inner workings of a cell, and revolutionized our understanding of the molecular mechanisms underlying physiological and disease states. In spite of these advances, however, visualization of certain classes of molecules (e.g. lipids) at the sub-cellular level has remained elusive. Recently developed chemical imaging modalities – Coherent Anti-Stokes Raman Scattering (CARS) microscopy and Stimulated Raman Scattering (SRS) microscopy – have helped bridge this gap. By selectively imaging the vibration of a specific chemical group, these non-invasive techniques allow high-resolution imaging of individual molecules in vivo, and circumvent the need for potentially perturbative extrinsic labels. These tools have already been applied to the study of fat metabolism, helping uncover novel regulators of lipid storage. Here we review the underlying principle of CARS and SRS microscopy, and discuss the advantages and caveats of each technique. We also review recent applications of these tools in the study of lipids as well as other biomolecules, and conclude with a brief guide for interested researchers to build and use CARS/SRS systems for their own research. PMID:24576891

Characterization of malaria infected blood cells by scanning confocal laser and acoustic vector contrast microscopy

NASA Astrophysics Data System (ADS)

Ahmed Mohamed, E. T.; Schubert, S.; Gilberger, T. W.; Kamanyi, A., Jr.; Wannemacher, R.; Grill, W.

2006-03-01

Acoustic and optical multiple contrast microscopy has been employed in order to explore characterizable parameters of red blood cells, including cells infected by the parasite Plasmodium falciparum, in order to investigate cellular modifications caused by the infection and to identify possible detection schemes for disease monitoring. Imaging schemes were based on fluorescence, optical transmission, optical reflection, and amplitude and phase of ultrasound reflected from the cells. Contrast variations observed in acoustic microscopy, but not in optical microscopy, were tentatively ascribed to changes caused by the infection.

The multi-modal Australian ScienceS Imaging and Visualization Environment (MASSIVE) high performance computing infrastructure: applications in neuroscience and neuroinformatics research

PubMed Central

Goscinski, Wojtek J.; McIntosh, Paul; Felzmann, Ulrich; Maksimenko, Anton; Hall, Christopher J.; Gureyev, Timur; Thompson, Darren; Janke, Andrew; Galloway, Graham; Killeen, Neil E. B.; Raniga, Parnesh; Kaluza, Owen; Ng, Amanda; Poudel, Govinda; Barnes, David G.; Nguyen, Toan; Bonnington, Paul; Egan, Gary F.

2014-01-01

The Multi-modal Australian ScienceS Imaging and Visualization Environment (MASSIVE) is a national imaging and visualization facility established by Monash University, the Australian Synchrotron, the Commonwealth Scientific Industrial Research Organization (CSIRO), and the Victorian Partnership for Advanced Computing (VPAC), with funding from the National Computational Infrastructure and the Victorian Government. The MASSIVE facility provides hardware, software, and expertise to drive research in the biomedical sciences, particularly advanced brain imaging research using synchrotron x-ray and infrared imaging, functional and structural magnetic resonance imaging (MRI), x-ray computer tomography (CT), electron microscopy and optical microscopy. The development of MASSIVE has been based on best practice in system integration methodologies, frameworks, and architectures. The facility has: (i) integrated multiple different neuroimaging analysis software components, (ii) enabled cross-platform and cross-modality integration of neuroinformatics tools, and (iii) brought together neuroimaging databases and analysis workflows. MASSIVE is now operational as a nationally distributed and integrated facility for neuroinfomatics and brain imaging research. PMID:24734019

Probing Cytoskeletal Structures by Coupling Optical Superresolution and AFM Techniques for a Correlative Approach

PubMed Central

Chacko, Jenu Varghese; Zanacchi, Francesca Cella; Diaspro, Alberto

2013-01-01

In this article, we describe and show the application of some of the most advanced fluorescence superresolution techniques, STED AFM and STORM AFM microscopy towards imaging of cytoskeletal structures, such as microtubule filaments. Mechanical and structural properties can play a relevant role in the investigation of cytoskeletal structures of interest, such as microtubules, that provide support to the cell structure. In fact, the mechanical properties, such as the local stiffness and the elasticity, can be investigated by AFM force spectroscopy with tens of nanometers resolution. Force curves can be analyzed in order to obtain the local elasticity (and the Young's modulus calculation by fitting the force curves from every pixel of interest), and the combination with STED/STORM microscopy integrates the measurement with high specificity and yields superresolution structural information. This hybrid modality of superresolution-AFM working is a clear example of correlative multimodal microscopy. PMID:24027190

High spatial resolution soft-x-ray microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyer-Ilse, W.; Medecki, H.; Brown, J.T.

1997-04-01

A new soft x-ray microscope (XM-1) with high spatial resolution has been constructed by the Center for X-ray Optics. It uses bending magnet radiation from beamline 6.1 at the Advanced Light Source, and is used in a variety of projects and applications in the life and physical sciences. Most of these projects are ongoing. The instrument uses zone plate lenses and achieves a resolution of 43 nm, measured over 10% to 90% intensity with a knife edge test sample. X-ray microscopy permits the imaging of relatively thick samples, up to 10 {mu}m thick, in water. XM-1 has an easy tomore » use interface, that utilizes visible light microscopy to precisely position and focus the specimen. The authors describe applications of this device in the biological sciences, as well as in studying industrial applications including structured polymer samples.« less

Frontiers of in situ electron microscopy

DOE PAGES

Zheng, Haimei; Zhu, Yimei; Meng, Shirley Ying

2015-01-01

In situ transmission electron microscopy (TEM) has become an increasingly important tool for materials characterization. It provides key information on the structural dynamics of a material during transformations and the correlation between structure and properties of materials. With the recent advances in instrumentation, including aberration corrected optics, sample environment control, the sample stage, and fast and sensitive data acquisition, in situ TEM characterization has become more and more powerful. In this article, a brief review of the current status and future opportunities of in situ TEM is included. It also provides an introduction to the six articles covered by inmore » this issue of MRS Bulletin explore the frontiers of in situ electron microscopy, including liquid and gas environmental TEM, dynamic four-dimensional TEM, nanomechanics, ferroelectric domain switching studied by in situ TEM, and state-of-the-art atomic imaging of light elements (i.e., carbon atoms) and individual defects.« less

A user's guide to localization-based super-resolution fluorescence imaging.

PubMed

Dempsey, Graham T

2013-01-01

Advances in far-field fluorescence microscopy over the past decade have led to the development of super-resolution imaging techniques that provide more than an order of magnitude improvement in spatial resolution compared to conventional light microscopy. One such approach, called Stochastic Optical Reconstruction Microscopy (STORM) uses the sequential, nanometer-scale localization of individual fluorophores to reconstruct a high-resolution image of a structure of interest. This is an attractive method for biological investigation at the nanoscale due to its relative simplicity, both conceptually and practically in the laboratory. Like most research tools, however, the devil is in the details. The aim of this chapter is to serve as a guide for applying STORM to the study of biological samples. This chapter will discuss considerations for choosing a photoswitchable fluorescent probe, preparing a sample, selecting hardware for data acquisition, and collecting and analyzing data for image reconstruction. Copyright © 2013 Elsevier Inc. All rights reserved.

Possibilities and limitations of advanced transmission electron microscopy for carbon-based nanomaterials

PubMed Central

Bittencourt, Carla; Van Tendeloo, Gustaaf

2015-01-01

Summary A major revolution for electron microscopy in the past decade is the introduction of aberration correction, which enables one to increase both the spatial resolution and the energy resolution to the optical limit. Aberration correction has contributed significantly to the imaging at low operating voltages. This is crucial for carbon-based nanomaterials which are sensitive to electron irradiation. The research of carbon nanomaterials and nanohybrids, in particular the fundamental understanding of defects and interfaces, can now be carried out in unprecedented detail by aberration-corrected transmission electron microscopy (AC-TEM). This review discusses new possibilities and limits of AC-TEM at low voltage, including the structural imaging at atomic resolution, in three dimensions and spectroscopic investigation of chemistry and bonding. In situ TEM of carbon-based nanomaterials is discussed and illustrated through recent reports with particular emphasis on the underlying physics of interactions between electrons and carbon atoms. PMID:26425406

Plasmon 3D Electron Tomography and Local Electric-Field Enhancement of Engineered Plasmonic Nanoantennas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Archanjo, B. S.; Vasconcelos, T. L.; Oliveira, B. S.

Plasmonic nano-antennas are pushing the limits of optical imaging resolution capabilities in near-field scanning optical microscopy (NSOM). Accordingly, these techniques are driving the basic understanding of photonic and optoelectronic nanoscale devices with applications in sensing, energy conversion, solid-state lighting and information technology. Imaging the localized surface plasmon resonance (LSPR) at the nanoscale is a key to understanding the optical responses of a given tip geometry in order to engineer better plasmonic nano-antennas for near-field experiments. In recent years the advancement of focused ion beam technology provides the ability to directly modify plasmonic structures with nanometer resolution. Also, scanning transmission electronmore » microscopy (STEM) with electron energy loss spectroscopy (EELS) is an established technique allowing imaging of LSPR. Specifically, the combination of these two techniques provides spectrally sensitive two-dimensional (2D) imaging information to better visualize and understand LSPR on the nanometer scale. This can be combined with electron tomography to provide the three-dimensional LSPR distribution. Here in this paper we demonstrate the fabrication of Au nano-pyramids using helium ion microscopy, and analyze the LSPR in 3D reconstructions produced by total variation (TV)-norm minimization of a set of 2D STEM-EELS maps. Additionally, a boundary element simulation method was used to verify the experimentally observed nanopyramid LSPR modes. Finally, we show that the point-spread-functions (PSF) of LSPR mode hot spots in nanopyramids differ to local electric-field enhancement under optical excitation making direct comparison to NSOM experimental resolution difficult. However, the STEM-EELS results show how LSPR modes are influenced by the tip characteristics, which can inform the development of new nano-antenna designs.« less

Plasmon 3D Electron Tomography and Local Electric-Field Enhancement of Engineered Plasmonic Nanoantennas

DOE PAGES

Archanjo, B. S.; Vasconcelos, T. L.; Oliveira, B. S.; ...

2018-06-01

Plasmonic nano-antennas are pushing the limits of optical imaging resolution capabilities in near-field scanning optical microscopy (NSOM). Accordingly, these techniques are driving the basic understanding of photonic and optoelectronic nanoscale devices with applications in sensing, energy conversion, solid-state lighting and information technology. Imaging the localized surface plasmon resonance (LSPR) at the nanoscale is a key to understanding the optical responses of a given tip geometry in order to engineer better plasmonic nano-antennas for near-field experiments. In recent years the advancement of focused ion beam technology provides the ability to directly modify plasmonic structures with nanometer resolution. Also, scanning transmission electronmore » microscopy (STEM) with electron energy loss spectroscopy (EELS) is an established technique allowing imaging of LSPR. Specifically, the combination of these two techniques provides spectrally sensitive two-dimensional (2D) imaging information to better visualize and understand LSPR on the nanometer scale. This can be combined with electron tomography to provide the three-dimensional LSPR distribution. Here in this paper we demonstrate the fabrication of Au nano-pyramids using helium ion microscopy, and analyze the LSPR in 3D reconstructions produced by total variation (TV)-norm minimization of a set of 2D STEM-EELS maps. Additionally, a boundary element simulation method was used to verify the experimentally observed nanopyramid LSPR modes. Finally, we show that the point-spread-functions (PSF) of LSPR mode hot spots in nanopyramids differ to local electric-field enhancement under optical excitation making direct comparison to NSOM experimental resolution difficult. However, the STEM-EELS results show how LSPR modes are influenced by the tip characteristics, which can inform the development of new nano-antenna designs.« less

Acousto-optical tunable filter for combined wideband, spectral, and optical coherence microscopy.

PubMed

Machikhin, Alexander S; Pozhar, Vitold E; Viskovatykh, Alexander V; Burmak, Ludmila I

2015-09-01

A multimodal technique for inspection of microscopic objects by means of wideband optical microscopy, spectral microscopy, and optical coherence microscopy is described, implemented, and tested. The key feature is the spectral selection of light in the output arm of an interferometer with use of the specialized imaging acousto-optical tunable filter. In this filter, two interfering optical beams are diffracted via the same ultrasound wave without destruction of interference image structure. The basic requirements for the acousto-optical tunable filter are defined, and mathematical formulas for calculation of its parameters are derived. Theoretical estimation of the achievable accuracy of the 3D image reconstruction is presented and experimental proofs are given. It is demonstrated that spectral imaging can also be accompanied by measurement of the quantitative reflectance spectra. Examples of inspection of optically transparent and nontransparent samples demonstrate the applicability of the technique.

Biobeam—Multiplexed wave-optical simulations of light-sheet microscopy

PubMed Central

Weigert, Martin; Bundschuh, Sebastian T.

2018-01-01

Sample-induced image-degradation remains an intricate wave-optical problem in light-sheet microscopy. Here we present biobeam, an open-source software package that enables simulation of operational light-sheet microscopes by combining data from 105–106 multiplexed and GPU-accelerated point-spread-function calculations. The wave-optical nature of these simulations leads to the faithful reproduction of spatially varying aberrations, diffraction artifacts, geometric image distortions, adaptive optics, and emergent wave-optical phenomena, and renders image-formation in light-sheet microscopy computationally tractable. PMID:29652879

Lensfree On-Chip Microscopy and Tomography for Bio-Medical Applications

PubMed Central

Isikman, Serhan O.; Bishara, Waheb; Mudanyali, Onur; Sencan, Ikbal; Su, Ting-Wei; Tseng, Derek; Yaglidere, Oguzhan; Sikora, Uzair; Ozcan, Aydogan

2012-01-01

Lensfree on-chip holographic microscopy is an emerging technique that offers imaging of biological specimens over a large field-of-view without using any lenses or bulky optical components. Lending itself to a compact, cost-effective and mechanically robust architecture, lensfree on-chip holographic microscopy can offer an alternative toolset addressing some of the emerging needs of microscopic analysis and diagnostics in low-resource settings, especially for telemedicine applications. In this review, we summarize the latest achievements in lensfree optical microscopy based on partially coherent on-chip holography, including portable telemedicine microscopy, cell-phone based microscopy and field-portable optical tomographic microscopy. We also discuss some of the future directions for telemedicine microscopy and its prospects to help combat various global health challenges. PMID:24478572

Three-dimensional holographic optical manipulation through a high-numerical-aperture soft-glass multimode fibre

NASA Astrophysics Data System (ADS)

Leite, Ivo T.; Turtaev, Sergey; Jiang, Xin; Šiler, Martin; Cuschieri, Alfred; Russell, Philip St. J.; Čižmár, Tomáš

2018-01-01

Holographic optical tweezers (HOT) hold great promise for many applications in biophotonics, allowing the creation and measurement of minuscule forces on biomolecules, molecular motors and cells. Geometries used in HOT currently rely on bulk optics, and their exploitation in vivo is compromised by the optically turbid nature of tissues. We present an alternative HOT approach in which multiple three-dimensional (3D) traps are introduced through a high-numerical-aperture multimode optical fibre, thus enabling an equally versatile means of manipulation through channels having cross-section comparable to the size of a single cell. Our work demonstrates real-time manipulation of 3D arrangements of micro-objects, as well as manipulation inside otherwise inaccessible cavities. We show that the traps can be formed over fibre lengths exceeding 100 mm and positioned with nanometric resolution. The results provide the basis for holographic manipulation and other high-numerical-aperture techniques, including advanced microscopy, through single-core-fibre endoscopes deep inside living tissues and other complex environments.

Far-field radially polarized focal spot from plasmonic spiral structure combined with central aperture antenna

PubMed Central

Mao, Lei; Ren, Yuan; Lu, Yonghua; Lei, Xinrui; Jiang, Kang; Li, Kuanguo; Wang, Yong; Cui, Chenjing; Wen, Xiaolei; Wang, Pei

2016-01-01

Manipulation of a vector micro-beam with an optical antenna has significant potentials for nano-optical technology applications including bio-optics, optical fabrication, and quantum information processing. We have designed and demonstrated a central aperture antenna within an Archimedean spiral that extracts the bonding plasmonic field from a surface to produce a new vector focal spot in far-field. The properties of this vector focal field are revealed by confocal microscopy and theoretical simulations. The pattern, polarization and phase of the focal field are determined by the incident light and by the chirality of the Archimedean spiral. For incident light with right-handed circular polarization, the left-handed spiral (one-order chirality) outputs a micro-radially polarized focal field. Our results reveal the relationship between the near-field and far-field distributions of the plasmonic spiral structure, and the structure has the potential to lead to advances in diverse applications such as plasmonic lenses, near-field angular momentum detection, and optical tweezers. PMID:27009383

Varifocal MOEMS fiber scanner for confocal endomicroscopy.

PubMed

Meinert, Tobias; Weber, Niklas; Zappe, Hans; Seifert, Andreas

2014-12-15

Based on an advanced silicon optical bench technology with integrated MOEMS (Micro-Opto-Electro-Mechanical-System) components, a piezo-driven fiber scanner for confocal microscopy has been developed. This highly-miniaturized technology allows integration into an endoscope with a total outer probe diameter of 2.5 mm. The system features a hydraulically-driven varifocal lens providing axial confocal scanning without any translational movement of components. The demonstrated resolutions are 1.7 μm laterally and 19 μm axially.

Single-Molecule Optical Spectroscopy and Imaging: From Early Steps to Recent Advances

NASA Astrophysics Data System (ADS)

Moerner, William E.

The initial steps toward optical detection and spectroscopy of single molecules arose out of the study of spectral hole-burning in inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989. In the early 1990s, many fascinating physical effects were observed for individual molecules such as spectral diffusion, optical switching, vibrational spectra, and magnetic resonance of a single molecular spin. Since the mid-1990s when experiments moved to room temperature, a wide variety of biophysical effects may be explored, and a number of physical phenomena from the low temperature studies have analogs at high temperature. Recent advances worldwide cover a huge range, from in vitro studies of enzymes, proteins, and oligonucleotides, to observations in real time of a single protein performing a specific function inside a living cell. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation of individual fluorophores leads naturally to localization beyond the optical diffraction limit. Combining this with active optical control of the number of emitting molecules leads to superresolution imaging, a new frontier for optical microscopy beyond the optical diffraction limit and for chemical design of photoswitchable fluorescent labels. Finally, to study one molecule in aqueous solution without surface perturbations, a new electrokinetic trap is described (the ABEL trap) which can trap single small biomolecules without the need for large dielectric beads.

Rotary-scanning optical resolution photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Qi, Weizhi; Xi, Lei

2016-10-01

Optical resolution photoacoustic microscopy (ORPAM) is currently one of the fastest evolving photoacoustic imaging modalities. It has a comparable spatial resolution to pure optical microscopic techniques such as epifluorescence microscopy, confocal microscopy, and two-photon microscopy, but also owns a deeper penetration depth. In this paper, we report a rotary-scanning (RS)-ORPAM that utilizes a galvanometer scanner integrated with objective to achieve rotary laser scanning. A 15 MHz cylindrically focused ultrasonic transducer is mounted onto a motorized rotation stage to follow optical scanning traces synchronously. To minimize the loss of signal to noise ratio, the acoustic focus is precisely adjusted to reach confocal with optical focus. Black tapes and carbon fibers are firstly imaged to evaluate the performance of the system, and then in vivo imaging of vasculature networks inside the ears and brains of mice is demonstrated using this system.

The connectomics challenge

PubMed Central

Silvestri, Ludovico; Sacconi, Leonardo; Pavone, Francesco Saverio

Summary One of the most fascinating challenges in neuroscience is the reconstruction of the connectivity map of the brain. Recent years have seen a rapid expansion in the field of connectomics, whose aim is to trace this map and understand its relationship with neural computation. Many different approaches, ranging from electron and optical microscopy to magnetic resonance imaging, have been proposed to address the connectomics challenge on various spatial scales and in different species. Here, we review the main technological advances in the microscopy techniques applied to connectomics, highlighting the potential and limitations of the different methods. Finally, we briefly discuss the role of connectomics in the Human Brain Project, the Future and Emerging Technologies (FET) Flagship recently approved by the European Commission. PMID:24139653

An approach to spin-resolved molecular gas microscopy

NASA Astrophysics Data System (ADS)

Covey, Jacob P.; De Marco, Luigi; Acevedo, Óscar L.; Rey, Ana Maria; Ye, Jun

2018-04-01

Ultracold polar molecules are an ideal platform for studying many-body physics with long-range dipolar interactions. Experiments in this field have progressed enormously, and several groups are pursuing advanced apparatus for manipulation of molecules with electric fields as well as single-atom-resolved in situ detection. Such detection has become ubiquitous for atoms in optical lattices and tweezer arrays, but has yet to be demonstrated for ultracold polar molecules. Here we present a proposal for the implementation of site-resolved microscopy for polar molecules, and specifically discuss a technique for spin-resolved molecular detection. We use numerical simulation of spin dynamics of lattice-confined polar molecules to show how such a scheme would be of utility in a spin-diffusion experiment.

In vivo measurements of cutaneous melanin across spatial scales: using multiphoton microscopy and spatial frequency domain spectroscopy.

PubMed

Saager, Rolf B; Balu, Mihaela; Crosignani, Viera; Sharif, Ata; Durkin, Anthony J; Kelly, Kristen M; Tromberg, Bruce J

2015-06-01

The combined use of nonlinear optical microscopy and broadband reflectance techniques to assess melanin concentration and distribution thickness in vivo over the full range of Fitzpatrick skin types is presented. Twelve patients were measured using multiphoton microscopy (MPM) and spatial frequency domain spectroscopy (SFDS) on both dorsal forearm and volar arm, which are generally sun-exposed and non-sun-exposed areas, respectively. Both MPM and SFDS measured melanin volume fractions between (skin type I non-sun-exposed) and 20% (skin type VI sun exposed). MPM measured epidermal (anatomical) thickness values ~30-65 μm, while SFDS measured melanin distribution thickness based on diffuse optical path length. There was a strong correlation between melanin concentration and melanin distribution (epidermal) thickness measurements obtained using the two techniques. While SFDS does not have the ability to match the spatial resolution of MPM, this study demonstrates that melanin content as quantified using SFDS is linearly correlated with epidermal melanin as measured using MPM (R² = 0.8895). SFDS melanin distribution thickness is correlated to MPM values (R² = 0.8131). These techniques can be used individually and/or in combination to advance our understanding and guide therapies for pigmentation-related conditions as well as light-based treatments across a full range of skin types.

Emerging enhanced imaging technologies of the esophagus: spectroscopy, confocal laser endomicroscopy, and optical coherence tomography.

PubMed

Robles, Lourdes Y; Singh, Satish; Fisichella, Piero Marco

2015-05-15

Despite advances in diagnoses and therapy, esophageal adenocarcinoma remains a highly lethal neoplasm. Hence, a great interest has been placed in detecting early lesions and in the detection of Barrett esophagus (BE). Advanced imaging technologies of the esophagus have then been developed with the aim of improving biopsy sensitivity and detection of preplastic and neoplastic cells. The purpose of this article was to review emerging imaging technologies for esophageal pathology, spectroscopy, confocal laser endomicroscopy (CLE), and optical coherence tomography (OCT). We conducted a PubMed search using the search string "esophagus or esophageal or oesophageal or oesophagus" and "Barrett or esophageal neoplasm" and "spectroscopy or optical spectroscopy" and "confocal laser endomicroscopy" and "confocal microscopy" and "optical coherence tomography." The first and senior author separately reviewed all articles. Our search identified: 19 in vivo studies with spectroscopy that accounted for 1021 patients and 4 ex vivo studies; 14 clinical CLE in vivo studies that accounted for 941 patients and 1 ex vivo study with 13 patients; and 17 clinical OCT in vivo studies that accounted for 773 patients and 2 ex vivo studies. Human studies using spectroscopy had a very high sensitivity and specificity for the detection of BE. CLE showed a high interobserver agreement in diagnosing esophageal pathology and an accuracy of predicting neoplasia. We also found several clinical studies that reported excellent diagnostic sensitivity and specificity for the detection of BE using OCT. Advanced imaging technology for the detection of esophageal lesions is a promising field that aims to improve the detection of early esophageal lesions. Although advancing imaging techniques improve diagnostic sensitivities and specificities, their integration into diagnostic protocols has yet to be perfected. Copyright © 2015 Elsevier Inc. All rights reserved.

Surface slope metrology of highly curved x-ray optics with an interferometric microscope

NASA Astrophysics Data System (ADS)

Gevorkyan, Gevork S.; Centers, Gary; Polonska, Kateryna S.; Nikitin, Sergey M.; Lacey, Ian; Yashchuk, Valeriy V.

2017-09-01

The development of deterministic polishing techniques has given rise to vendors that manufacture high quality threedimensional x-ray optics. The surface metrology on these optics remains a difficult task. For the fabrication, vendors usually use unique surface metrology tools, generally developed on site, that are not available in the optical metrology labs at x-ray facilities. At the Advanced Light Source X-Ray Optics Laboratory, we have developed a rather straightforward interferometric-microscopy-based procedure capable of sub microradian characterization of sagittal slope variation of x-ray optics for two-dimensionally focusing and collimating (such as ellipsoids, paraboloids, etc.). In the paper, we provide the mathematical foundation of the procedure and describe the related instrument calibration. We also present analytical expression describing the ideal surface shape in the sagittal direction of a spheroid specified by the conjugate parameters of the optic's beamline application. The expression is useful when analyzing data obtained with such optics. The high efficiency of the developed measurement and data analysis procedures is demonstrated in results of measurements with a number of x-ray optics with sagittal radius of curvature between 56 mm and 480 mm. We also discuss potential areas of further improvement.

Bending the Rules: Widefield Microscopy and the Abbe Limit of Resolution

PubMed Central

Verdaasdonk, Jolien S.; Stephens, Andrew D.; Haase, Julian; Bloom, Kerry

2014-01-01

One of the most fundamental concepts of microscopy is that of resolution–the ability to clearly distinguish two objects as separate. Recent advances such as structured illumination microscopy (SIM) and point localization techniques including photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM) strive to overcome the inherent limits of resolution of the modern light microscope. These techniques, however, are not always feasible or optimal for live cell imaging. Thus, in this review, we explore three techniques for extracting high resolution data from images acquired on a widefield microscope–deconvolution, model convolution, and Gaussian fitting. Deconvolution is a powerful tool for restoring a blurred image using knowledge of the point spread function (PSF) describing the blurring of light by the microscope, although care must be taken to ensure accuracy of subsequent quantitative analysis. The process of model convolution also requires knowledge of the PSF to blur a simulated image which can then be compared to the experimentally acquired data to reach conclusions regarding its geometry and fluorophore distribution. Gaussian fitting is the basis for point localization microscopy, and can also be applied to tracking spot motion over time or measuring spot shape and size. All together, these three methods serve as powerful tools for high-resolution imaging using widefield microscopy. PMID:23893718

Predicting scattering scanning near-field optical microscopy of mass-produced plasmonic devices

NASA Astrophysics Data System (ADS)

Otto, Lauren M.; Burgos, Stanley P.; Staffaroni, Matteo; Ren, Shen; Süzer, Özgün; Stipe, Barry C.; Ashby, Paul D.; Hammack, Aeron T.

2018-05-01

Scattering scanning near-field optical microscopy enables optical imaging and characterization of plasmonic devices with nanometer-scale resolution well below the diffraction limit. This technique enables developers to probe and understand the waveguide-coupled plasmonic antenna in as-fabricated heat-assisted magnetic recording heads. In order to validate and predict results and to extract information from experimental measurements that is physically comparable to simulations, a model was developed to translate the simulated electric field into expected near-field measurements using physical parameters specific to scattering scanning near-field optical microscopy physics. The methods used in this paper prove that scattering scanning near-field optical microscopy can be used to determine critical sub-diffraction-limited dimensions of optical field confinement, which is a crucial metrology requirement for the future of nano-optics, semiconductor photonic devices, and biological sensing where the near-field character of light is fundamental to device operation.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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.

Stochastic Optical Reconstruction Microscopy (STORM).

PubMed

Xu, Jianquan; Ma, Hongqiang; Liu, Yang

2017-07-05

Super-resolution (SR) fluorescence microscopy, a class of optical microscopy techniques at a spatial resolution below the diffraction limit, has revolutionized the way we study biology, as recognized by the Nobel Prize in Chemistry in 2014. Stochastic optical reconstruction microscopy (STORM), a widely used SR technique, is based on the principle of single molecule localization. STORM routinely achieves a spatial resolution of 20 to 30 nm, a ten-fold improvement compared to conventional optical microscopy. Among all SR techniques, STORM offers a high spatial resolution with simple optical instrumentation and standard organic fluorescent dyes, but it is also prone to image artifacts and degraded image resolution due to improper sample preparation or imaging conditions. It requires careful optimization of all three aspects-sample preparation, image acquisition, and image reconstruction-to ensure a high-quality STORM image, which will be extensively discussed in this unit. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

Special issue on high-resolution optical imaging

NASA Astrophysics Data System (ADS)

Smith, Peter J. S.; Davis, Ilan; Galbraith, Catherine G.; Stemmer, Andreas

2013-09-01

The pace of development in the field of advanced microscopy is truly breath-taking, and is leading to major breakthroughs in our understanding of molecular machines and cell function. This special issue of Journal of Optics draws attention to a number of interesting approaches, ranging from fluorescence and imaging of unlabelled cells, to computational methods, all of which are describing the ever increasing detail of the dynamic behaviour of molecules in the living cell. This is a field which traditionally, and currently, demonstrates a marvellous interplay between the disciplines of physics, chemistry and biology, where apparent boundaries to resolution dissolve and living cells are viewed in ever more clarity. It is fertile ground for those interested in optics and non-conventional imaging to contribute high-impact outputs in the fields of cell biology and biomedicine. The series of articles presented here has been selected to demonstrate this interdisciplinarity and to encourage all those with a background in the physical sciences to 'dip their toes' into the exciting and dynamic discoveries surrounding cell function. Although single molecule super-resolution microscopy is commercially available, specimen preparation and interpretation of single molecule data remain a major challenge for scientists wanting to adopt the techniques. The paper by Allen and Davidson [1] provides a much needed detailed introduction to the practical aspects of stochastic optical reconstruction microscopy, including sample preparation, image acquisition and image analysis, as well as a brief description of the different variants of single molecule localization microscopy. Since super-resolution microscopy is no longer restricted to three-dimensional imaging of fixed samples, the review by Fiolka [2] is a timely introduction to techniques that have been successfully applied to four-dimensional live cell super-resolution microscopy. The combination of multiple high-resolution techniques, such as the combination of light sheet and structured illumination microscopy (SIM), which efficiently utilize photon budget and avoid illuminating regions of the specimen not currently being imaged, hold the greatest promise for future biological applications. Therefore, the combined setup for SIM and single molecule localization microscopy (SMLM) described by Rossberger et al [3] will be very helpful and stimulating to advanced microscopists in further modifying their setups. The SIM image helps in identifying artefacts in SMLM reconstruction, e.g. when two active fluorophores are close together and get rejected as 'out-of-focus'. This combined setup is another way to facilitate imaging live samples. The article by Thomas et al [4] presents another advance for biological super-resolution imaging with a new approach to reconstruct optically sectioned images using structured illumination. The method produces images with higher spatial resolution and greater signal to noise compared to existing approaches. This algorithm demonstrates great promise for reconstructing biological images where the signal intensities are inherently lower. Shevchuk et al [5] present a non-optic near field approach to imaging with a review of scanning ion-conductance microscopy. This is a powerful alternative approach for examining the surface dynamics of living cells including exo and endocytosis, unlabelled, and at the level of the single event. Here they present the first data on combining this approach with fluorescence confocal microscopy—adding that extra dimension. Different approaches to label-free live cell imaging are presented in the papers by Patel et al [6], Mehta and Oldenbourg [7], as well as Rogers and Zheludev [8]. All three papers bring home the excitement of looking at live cell dynamics without reporters—Patel et al [6] review both the potential of coherent anti-Stokes Raman scattering and biological applications, where specific biomolecules are detected on the basis of their biophysical properties. Polarized light microscopy as presented by Mehta and Oldenbourg [7], describe a novel implementation of this technology to detect dichroism, and demonstrate beautifully its use in imaging unlabelled microtubules, mitochondria and lipid droplets. Sub-wavelength light focusing provides another avenue to super-resolution, and this is presented by Rogers and Zheludev [8]. Speculating on further improvements, these authors expect a resolution of 0.15λ. To date, the method has not been applied to low contrast, squishy and motile biotargets, but is included here for the clear potential to drive label-free imaging in new directions. A similar logic lies behind the inclusion of Parsons et al [9] where ultraviolet coherent diffractive imaging is further developed. These authors have demonstrated a shrink-wrap technique which reduces the integration time by a factor of 5, bringing closer the time when we have lab based imaging systems based on extreme ultraviolet and soft x-ray sources using sophisticated phase retrieval algorithms. Real biological specimens have spatially varying refractive indices that inevitably lead to aberrations and image distortions. Global refractive index matching of the embedding medium has been an historic solution, but unfortunately is not practical for live cell imaging. Adaptive optics appears an attractive solution and Simmonds and Booth [10] demonstrate the theoretical benefits of applying several adaptive optical elements, placed in different conjugate planes, to create a kind of 'inverse specimen' that unwarps phase distortions of the sample—but these have yet to be tested on real specimens. A difficulty in single molecule localization microscopy has been the determination of whether or not two molecules are colocalized. Kim et al [11] present a method for correcting bleed-through during multi-colour, single molecule localization microscopy. Such methods are welcome standards when trying to quantifiably interpret how close two molecules actually are. Rees et al [12] provide an invaluable overview of key image processing steps in localization microscopy. This paper is an excellent starting point for anyone implementing localization algorithms and the Matlab software provided will be invaluable; a strong paper on which to conclude our overview of the excellent articles brought together in this issue. One aspect brought home in several of these articles is the volume of data now being collected by high resolution live cell imaging. Data processing and image reconstruction will continue to be pressure points in the further development of instrumentation and analyses. We would hope that the series of papers presented here will motivate software engineers, optical physicists and biologists to contribute to the further development of this exciting field. References [1] Allen J R et al 2013 J. Opt. 15 094001 [2] Fiolka R et al 2013 J. Opt. 15 094002 [3] Rossberger S et al 2013 J. Opt. 15 094003 [4] Thomas B et al 2013 J. Opt. 15 094004 [5] Shevchuk A et al 2013 J. Opt. 15 094005 [6] Patel I et al 2013 J. Opt. 15 094006 [7] Mehta S B et al 2013 J. Opt. 15 094007 [8] Rogers E T F et al 2013 J. Opt. 15 094008 [9] Parsons A D et al 2013 J. Opt. 15 094009 [10] Simmonds R et al 2013 J. Opt. 15 094010 [11] Kim D et al 2013 J. Opt. 15 094011 [12] Rees E J et al 2013 J. Opt. 15 094012

LED-based interference-reflection microscopy combined with optical tweezers for quantitative three-dimensional microtubule imaging.

PubMed

Simmert, Steve; Abdosamadi, Mohammad Kazem; Hermsdorf, Gero; Schäffer, Erik

2018-05-28

Optical tweezers combined with various microscopy techniques are a versatile tool for single-molecule force spectroscopy. However, some combinations may compromise measurements. Here, we combined optical tweezers with total-internal-reflection-fluorescence (TIRF) and interference-reflection microscopy (IRM). Using a light-emitting diode (LED) for IRM illumination, we show that single microtubules can be imaged with high contrast. Furthermore, we converted the IRM interference pattern of an upward bent microtubule to its three-dimensional (3D) profile calibrated against the optical tweezers and evanescent TIRF field. In general, LED-based IRM is a powerful method for high-contrast 3D microscopy.

Symposium on Automation, Robotics and Advanced Computing for the National Space Program (2nd) Held in Arlington, Virginia on 9-11 March 1987

DTIC Science & Technology

1988-02-28

enormous investment in software. This is an area extremely important objective. We need additional where better methodologies , tools and theories...microscopy (SEM) and optical mi- [131 Hanson, A., et a. "A Methodology for the Develop- croscopy. Current activities include the study of SEM im- ment...through a phased knowledge engineering methodology Center (ARC) and NASA Johnson Space Center consisting of: prototype knowledge base develop- iJSC

Manfred Girbardt and Charles Bracker: outstanding pioneers in fungal microscopy.

PubMed

Bartnicki-Garcia, Salomon

2015-01-01

Midway through the twentieth century, the availability of new and improved optical and electronic microscopes facilitated rapid advances in the elucidation of the fine structure of fungal cells. In this Essay, I pay tribute to Manfred Girbardt (1919-1991) and Charles Bracker (1938-2012)—two individuals who, despite being separated by geography and the restrictions of the Cold War, both made equally fundamental discoveries in fungal cell ultrastructure and set high standards for specimen manipulation and image processing.

Optical biopsy of pre-malignant or degenerative lesions: the role of the inflammatory process

NASA Astrophysics Data System (ADS)

da Silva Martinho, Herculano

2011-03-01

Recent technological advances in fiber optics, light sources, detectors, and molecular biology have stimulated unprecedented development of optical methods to detect pathological changes in tissues. These methods, collectively termed "optical biopsy," are nondestructive in situ and real-time assays. Optical biopsy techniques as fluorescence spectroscopy, polarized light scattering spectroscopy, optical coherence tomography, confocal reflectance microscopy, and Raman spectroscopy had been extensively used to characterize several pathological tissues. In special, Raman spectroscopy technique had been able to probe several biochemical alterations due to pathology development as change in the DNA, glycogen, phospholipid, non-collagenous proteins. All studies claimed that the optical biopsy methods were able to discriminate normal and malignant tissues. However, few studies had been devoted to the discrimination of very common subtle or early pathological states as inflammatory process, which are always present on, e.g., cancer lesion border. In this work we present a systematic comparison of optical biopsy data on several kinds of lesions were inflammatory infiltrates play the role (breast, cervical, and oral lesion). It will be discussed the essential conditions for the optimization of discrimination among normal and alterated states based on statistical analysis.

Rapid prototyping of Fresnel zone plates via direct Ga(+) ion beam lithography for high-resolution X-ray imaging.

PubMed

Keskinbora, Kahraman; Grévent, Corinne; Eigenthaler, Ulrike; Weigand, Markus; Schütz, Gisela

2013-11-26

A significant challenge to the wide utilization of X-ray microscopy lies in the difficulty in fabricating adequate high-resolution optics. To date, electron beam lithography has been the dominant technique for the fabrication of diffractive focusing optics called Fresnel zone plates (FZP), even though this preparation method is usually very complicated and is composed of many fabrication steps. In this work, we demonstrate an alternative method that allows the direct, simple, and fast fabrication of FZPs using focused Ga(+) beam lithography practically, in a single step. This method enabled us to prepare a high-resolution FZP in less than 13 min. The performance of the FZP was evaluated in a scanning transmission soft X-ray microscope where nanostructures as small as sub-29 nm in width were clearly resolved, with an ultimate cutoff resolution of 24.25 nm, demonstrating the highest first-order resolution for any FZP fabricated by the ion beam lithography technique. This rapid and simple fabrication scheme illustrates the capabilities and the potential of direct ion beam lithography (IBL) and is expected to increase the accessibility of high-resolution optics to a wider community of researchers working on soft X-ray and extreme ultraviolet microscopy using synchrotron radiation and advanced laboratory sources.

Detection, mapping, and quantification of single walled carbon nanotubes in histological specimens with photoacoustic microscopy.

PubMed

Avti, Pramod K; Hu, Song; Favazza, Christopher; Mikos, Antonios G; Jansen, John A; Shroyer, Kenneth R; Wang, Lihong V; Sitharaman, Balaji

2012-01-01

In the present study, the efficacy of multi-scale photoacoustic microscopy (PAM) was investigated to detect, map, and quantify trace amounts [nanograms (ng) to micrograms (µg)] of SWCNTs in a variety of histological tissue specimens consisting of cancer and benign tissue biopsies (histological specimens from implanted tissue engineering scaffolds). Optical-resolution (OR) and acoustic-resolution (AR)--Photoacoustic microscopy (PAM) was employed to detect, map and quantify the SWCNTs in a variety of tissue histological specimens and compared with other optical techniques (bright-field optical microscopy, Raman microscopy, near infrared (NIR) fluorescence microscopy). Both optical-resolution and acoustic-resolution PAM, allow the detection and quantification of SWCNTs in histological specimens with scalable spatial resolution and depth penetration. The noise-equivalent detection sensitivity to SWCNTs in the specimens was calculated to be as low as ∼7 pg. Image processing analysis further allowed the mapping, distribution, and quantification of the SWCNTs in the histological sections. The results demonstrate the potential of PAM as a promising imaging technique to detect, map, and quantify SWCNTs in histological specimens, and could complement the capabilities of current optical and electron microscopy techniques in the analysis of histological specimens containing SWCNTs.

Microsphere-aided optical microscopy and its applications for super-resolution imaging

NASA Astrophysics Data System (ADS)

Upputuri, Paul Kumar; Pramanik, Manojit

2017-12-01

The spatial resolution of a standard optical microscope (SOM) is limited by diffraction. In visible spectrum, SOM can provide ∼ 200 nm resolution. To break the diffraction limit several approaches were developed including scanning near field microscopy, metamaterial super-lenses, nanoscale solid immersion lenses, super-oscillatory lenses, confocal fluorescence microscopy, techniques that exploit non-linear response of fluorophores like stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, etc. Recently, photonic nanojet generated by a dielectric microsphere was used to break the diffraction limit. The microsphere-approach is simple, cost-effective and can be implemented under a standard microscope, hence it has gained enormous attention for super-resolution imaging. In this article, we briefly review the microsphere approach and its applications for super-resolution imaging in various optical imaging modalities.

Automated analysis of individual sperm cells using stain-free interferometric phase microscopy and machine learning.

PubMed

Mirsky, Simcha K; Barnea, Itay; Levi, Mattan; Greenspan, Hayit; Shaked, Natan T

2017-09-01

Currently, the delicate process of selecting sperm cells to be used for in vitro fertilization (IVF) is still based on the subjective, qualitative analysis of experienced clinicians using non-quantitative optical microscopy techniques. In this work, a method was developed for the automated analysis of sperm cells based on the quantitative phase maps acquired through use of interferometric phase microscopy (IPM). Over 1,400 human sperm cells from 8 donors were imaged using IPM, and an algorithm was designed to digitally isolate sperm cell heads from the quantitative phase maps while taking into consideration both the cell 3D morphology and contents, as well as acquire features describing sperm head morphology. A subset of these features was used to train a support vector machine (SVM) classifier to automatically classify sperm of good and bad morphology. The SVM achieves an area under the receiver operating characteristic curve of 88.59% and an area under the precision-recall curve of 88.67%, as well as precisions of 90% or higher. We believe that our automatic analysis can become the basis for objective and automatic sperm cell selection in IVF. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

New histopathologic and ultrastructural findings in Reis-Bücklers corneal dystrophy caused by the Arg124Leu mutation of TGFBI gene.

PubMed

Qiu, Wen-Ya; Zheng, Li-Bin; Pan, Fei; Wang, Bei-Bei; Yao, Yu-Feng

2016-09-02

Reis-Bücklers corneal dystrophy (RBCD) was consistently reported as a corneal dystrophy only affected Bowman's layer and superficial corneal stroma, and superficial keratectomy was a recommendation surgery for treatment in literatures. The study reported new histopathological and ultrastructural findings in RBCD caused by the Arg124Leu mutation of transforming growth factor induced (TGFBI) gene in a four-generation Chinese pedigree. Subjects including eight patients and seven unaffected family members received slit-lamp biomicroscopy and photography. DNA was obtained from all subjects, and exons 4 and 11 to 14 of TGFBI gene were analyzed by polymerase chain reaction and the products were sequenced. Anterior segment optical coherence tomography (AS OCT) and in vivo confocal microscopy were conducted for ten eyes of five patients. Based on the results of AS OCT and in vivo confocal microscopy, deep anterior lamellar keratoplasty (DLKP) using cryopreserved donor cornea was applied for four eyes of four patients. Four lamellar dystrophic corneal buttons were studied by light and transmission electron microscopy, and TGFBI immunohistochemistry. Eight patients had typical clinical manifestations of RBCD presenting recurrent painful corneal erosion starting in their early first decades, along with age-dependent progressive geographic corneal opacities. TGFBI sequencing revealed a heterozygous mutation, Arg124Leu in all eight patients. Anterior segment optical coherence tomography and in vivo confocal microscopy showed the dystrophic deposits involved not only in subepithelial and superficial stroma, but also in mid- or posterior stroma in four examined advanced eyes. Light microscopy showed Bowman's layer was absent, replaced by abnormal deposits stain bright red with Masson's trichrome. In superficial cornea, the deposits stacked and produced three to five continuous bands parallel to the corneal collagen lamellae. In mid- to posterior stroma, numerous granular or dot- like aggregates were heavily scattered, and most of them presented around the nuclei of stromal keratocytes. Transmission electron microscopy revealed the multiple electron-dense rod-shaped deposits aggregated and formed a characteristic pattern of three to five continuous bands in superficial cornea, which were similar to those seen under light microscopy. In mid- to posterior stroma, clusters of rod-shaped bodies were scattered extracellular or intracellular of the stromal keratocytes between the stromal lamellae suggesting the close relationship between mutated proteins and keratocyte. The study offer evidences indicating DLKP is a viable treatment option for advanced RBCD to avoid recurrence, and the mutated TGFBIp in dystrophic corneas are of keratocytes origin.

Array microscopy technology and its application to digital detection of Mycobacterium tuberculosis

NASA Astrophysics Data System (ADS)

McCall, Brian P.

Tuberculosis causes more deaths worldwide than any other curable infectious disease. This is the case despite tuberculosis appearing to be on the verge of eradication midway through the last century. Efforts at reversing the spread of tuberculosis have intensified since the early 1990s. Since then, microscopy has been the primary frontline diagnostic. In this dissertation, advances in clinical microscopy towards array microscopy for digital detection of Mycobacterium tuberculosis are presented. Digital array microscopy separates the tasks of microscope operation and pathogen detection and will reduce the specialization needed in order to operate the microscope. Distributing the work and reducing specialization will allow this technology to be deployed at the point of care, taking the front-line diagnostic for tuberculosis from the microscopy center to the community health center. By improving access to microscopy centers, hundreds of thousands of lives can be saved. For this dissertation, a lens was designed that can be manufactured as 4x6 array of microscopes. This lens design is diffraction limited, having less than 0.071 waves of aberration (root mean square) over the entire field of view. A total area imaged onto a full-frame digital image sensor is expected to be 3.94 mm2, which according to tuberculosis microscopy guidelines is more than sufficient for a sensitive diagnosis. The design is tolerant to single point diamond turning manufacturing errors, as found by tolerance analysis and by fabricating a prototype. Diamond micro-milling, a fabrication technique for lens array molds, was applied to plastic plano-concave and plano-convex lens arrays, and found to produce high quality optical surfaces. The micro-milling technique did not prove robust enough to produce bi-convex and meniscus lens arrays in a variety of lens shapes, however, and it required lengthy fabrication times. In order to rapidly prototype new lenses, a new diamond machining technique was developed called 4-axis single point diamond machining. This technique is 2-10x faster than micro-milling, depending on how advanced the micro-milling equipment is. With array microscope fabrication still in development, a single prototype of the lens designed for an array microscope was fabricated using single point diamond turning. The prototype microscope objective was validated in a pre-clinical trial. The prototype was compared with a standard clinical microscope objective in diagnostic tests. High concordance, a Fleiss's kappa of 0.88, was found between diagnoses made using the prototype and standard microscope objectives and a reference test. With the lens designed and validated and an advanced fabrication process developed, array microscopy technology is advanced to the point where it is feasible to rapidly prototype an array microscope for detection of tuberculosis and translate array microscope from an innovative concept to a device that can save lives.

Thermal and Optical Modulation of the Carrier Mobility in OTFTs Based on an Azo-anthracene Liquid Crystal Organic Semiconductor.

PubMed

Chen, Yantong; Li, Chao; Xu, Xiuru; Liu, Ming; He, Yaowu; Murtaza, Imran; Zhang, Dongwei; Yao, Chao; Wang, Yongfeng; Meng, Hong

2017-03-01

One of the most striking features of organic semiconductors compared with their corresponding inorganic counterparts is their molecular diversity. The major challenge in organic semiconductor material technology is creating molecular structural motifs to develop multifunctional materials in order to achieve the desired functionalities yet to optimize the specific device performance. Azo-compounds, because of their special photoresponsive property, have attracted extensive interest in photonic and optoelectronic applications; if incorporated wisely in the organic semiconductor groups, they can be innovatively utilized in advanced smart electronic applications, where thermal and photo modulation is applied to tune the electronic properties. On the basis of this aspiration, a novel azo-functionalized liquid crystal semiconductor material, (E)-1-(4-(anthracen-2-yl)phenyl)-2-(4-(decyloxy)phenyl)diazene (APDPD), is designed and synthesized for application in organic thin-film transistors (OTFTs). The UV-vis spectra of APDPD exhibit reversible photoisomerizaton upon photoexcitation, and the thin films of APDPD show a long-range orientational order based on its liquid crystal phase. The performance of OTFTs based on this material as well as the effects of thermal treatment and UV-irradiation on mobility are investigated. The molecular structure, stability of the material, and morphology of the thin films are characterized by thermal gravimetric analysis (TGA), polarizing optical microscopy (POM), (differential scanning calorimetry (DSC), UV-vis spectroscopy, atomic force microscopy (AFM), and scanning tunneling microscopy (STM). This study reveals that our new material has the potential to be applied in optical sensors, memories, logic circuits, and functional switches.

Trends in fluorescence imaging and related techniques to unravel biological information.

PubMed

Haustein, Elke; Schwille, Petra

2007-09-01

Optical microscopy is among the most powerful tools that the physical sciences have ever provided biology. It is indispensable for basic lab work, as well as for cutting edge research, as the visual monitoring of life processes still belongs to the most compelling evidences for a multitude of biomedical applications. Along with the rapid development of new probes and methods for the analysis of laser induced fluorescence, optical microscopy over past years experienced a vast increase of both new techniques and novel combinations of established methods to study biological processes with unprecedented spatial and temporal precision. On the one hand, major technical advances have significantly improved spatial resolution. On the other hand, life scientists are moving toward three- and even four-dimensional cell biology and biophysics involving time as a crucial coordinate to quantitatively understand living specimen. Monitoring the whole cell or tissue in real time, rather than producing snap-shot-like two-dimensional projections, will enable more physiological and, thus, more clinically relevant experiments, whereas an increase in temporal resolution facilitates monitoring fast nonperiodic processes as well as the quantitative analysis of characteristic dynamics.

Trends in fluorescence imaging and related techniques to unravel biological information

PubMed Central

Haustein, Elke; Schwille, Petra

2007-01-01

Optical microscopy is among the most powerful tools that the physical sciences have ever provided biology. It is indispensable for basic lab work, as well as for cutting edge research, as the visual monitoring of life processes still belongs to the most compelling evidences for a multitude of biomedical applications. Along with the rapid development of new probes and methods for the analysis of laser induced fluorescence, optical microscopy over past years experienced a vast increase of both new techniques and novel combinations of established methods to study biological processes with unprecedented spatial and temporal precision. On the one hand, major technical advances have significantly improved spatial resolution. On the other hand, life scientists are moving toward three- and even four-dimensional cell biology and biophysics involving time as a crucial coordinate to quantitatively understand living specimen. Monitoring the whole cell or tissue in real time, rather than producing snap-shot-like two-dimensional projections, will enable more physiological and, thus, more clinically relevant experiments, whereas an increase in temporal resolution facilitates monitoring fast nonperiodic processes as well as the quantitative analysis of characteristic dynamics. PMID:19404444

Raman spectroscopy and atomic force microscopy study of interfacial polytypism in GaP/Ge(111) heterostructures

NASA Astrophysics Data System (ADS)

Aggarwal, R.; Ingale, Alka A.; Dixit, V. K.

2018-01-01

Effects of lattice and polar/nonpolar mismatch between the GaP layer and Ge(111) substrate are investigated by spatially resolved Raman spectroscopy. The red shifted transverse optical (TO) and longitudinal optical (LO) phonons due to residual strain, along with asymmetry to TO phonon ∼358 cm-1 are observed in GaP/Ge(111). The peak intensity variation of mode ∼358 cm-1 with respect to TO phonon across the crystallographic morphed surface of GaP micro structures is associated with the topographical variations using atomic force microscopy mapping and Raman spectroscopy performed on both in plane and cross-sectional surface. Co-existence of GaP allotropes, i.e. wurtzite phase near heterojunction interface and dominant zinc-blende phase near surface is established using the spatially resolved polarized Raman spectroscopy from the cross sectional surface of heterostructures. This consistently explains effect of surface morphology on Raman spectroscopy from GaP(111). The study shows the way to identify crystalline phases in other advanced semiconductor heterostructures without any specific sample preparation.

Reflective small angle electron scattering to characterize nanostructures on opaque substrates

NASA Astrophysics Data System (ADS)

Friedman, Lawrence H.; Wu, Wen-Li; Fu, Wei-En; Chien, Yunsan

2017-09-01

Feature sizes in integrated circuits (ICs) are often at the scale of 10 nm and are ever shrinking. ICs appearing in today's computers and hand held devices are perhaps the most prominent examples. These smaller feature sizes demand equivalent advances in fast and accurate dimensional metrology for both development and manufacturing. Techniques in use and continuing to be developed include X-ray based techniques, optical scattering, and of course the electron and scanning probe microscopy techniques. Each of these techniques has their advantages and limitations. Here, the use of small angle electron beam scattering measurements in a reflection mode (RSAES) to characterize the dimensions and the shape of nanostructures on flat and opaque substrates is demonstrated using both experimental and theoretical evidence. In RSAES, focused electrons are scattered at angles smaller than 1 ° with the assistance of electron optics typically used in transmission electron microscopy. A proof-of-concept experiment is combined with rigorous electron reflection simulations to demonstrate the efficiency and accuracy of RSAES as a method of non-destructive measurement of shapes of features less than 10 nm in size on flat and opaque substrates.

Reflective Small Angle Electron Scattering to Characterize Nanostructures on Opaque Substrates.

PubMed

Friedman, Lawrence H; Wu, Wen-Li; Fu, Wei-En; Chien, Yunsan

2017-09-01

Features sizes in integrated circuits (ICs) are often at the scale of 10 nm and are ever shrinking. ICs appearing in today's computers and hand held devices are perhaps the most prominent examples. These smaller feature sizes demand equivalent advances in fast and accurate dimensional metrology for both development and manufacturing. Techniques in use and continuing to be developed include X-ray based techniques, optical scattering and of course the electron and scanning probe microscopy techniques. Each of these techniques have their advantages and limitations. Here the use of small angle electron beam scattering measurements in a reflection mode (RSAES) to characterize the dimensions and the shape of nanostructures on flat and opaque substrates is demonstrated using both experimental and theoretical evidence. In RSAES, focused electrons are scattered at angles smaller than 1° with the assistance of electron optics typically used in transmission electron microscopy. A proof-of-concept experiment is combined with rigorous electron reflection simulations to demonstrate the efficiency and accuracy of RSAES as a method of non-destructive measurement of shapes of features less than 10 nm in size on flat and opaque substrates.

Exploring photoinactivation of microbial biofilms using laser scanning microscopy and confined two-photon excitation.

PubMed

Thomsen, Hanna; Graf, Fabrice E; Farewell, Anne; Ericson, Marica B

2018-05-21

One pertinent complication in bacterial infection is the growth of biofilms, i.e., communities of surface-adhered bacteria resilient to antibiotics. Photodynamic inactivation has been proposed as an alternative to antibiotic treatment; however, novel techniques complementing standard efficacy measures are required. Herein, we present an approach employing multiphoton microscopy complemented with Airyscan super-resolution microscopy, to visualize the distribution of curcumin in Staphylococcus epidermidis biofilms. The effects of complexation of curcumin with hydroxypropyl-γ-cyclodextrin (HPγCD) were studied. It was shown that HPγCD-curcumin demonstrated higher bioavailability in the biofilms compared to curcumin, without affecting the subcellular uptake. Spectral quantification following photodynamic inactivation demonstrates a method for monitoring elimination of biofilms in real time using noninvasive 3D imaging. Additionally, spatially confined two-photon inactivation was demonstrated for the first time in biofilms. These results support the feasibility of advanced optical microscopy as a sensitive tool for evaluating treatment efficacy in biofilms towards improved mechanistic studies of photodynamic inactivation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Quantitative Aspects of Single Molecule Microscopy

PubMed Central

Ober, Raimund J.; Tahmasbi, Amir; Ram, Sripad; Lin, Zhiping; Ward, E. Sally

2015-01-01

Single molecule microscopy is a relatively new optical microscopy technique that allows the detection of individual molecules such as proteins in a cellular context. This technique has generated significant interest among biologists, biophysicists and biochemists, as it holds the promise to provide novel insights into subcellular processes and structures that otherwise cannot be gained through traditional experimental approaches. Single molecule experiments place stringent demands on experimental and algorithmic tools due to the low signal levels and the presence of significant extraneous noise sources. Consequently, this has necessitated the use of advanced statistical signal and image processing techniques for the design and analysis of single molecule experiments. In this tutorial paper, we provide an overview of single molecule microscopy from early works to current applications and challenges. Specific emphasis will be on the quantitative aspects of this imaging modality, in particular single molecule localization and resolvability, which will be discussed from an information theoretic perspective. We review the stochastic framework for image formation, different types of estimation techniques and expressions for the Fisher information matrix. We also discuss several open problems in the field that demand highly non-trivial signal processing algorithms. PMID:26167102

High-resolution imaging of the central nervous system: how novel imaging methods combined with navigation strategies will advance patient care.

PubMed

Farooq, Hamza; Genis, Helen; Alarcon, Joseph; Vuong, Barry; Jivraj, Jamil; Yang, Victor X D; Cohen-Adad, Julien; Fehlings, Michael G; Cadotte, David W

2015-01-01

This narrative review captures a subset of recent advances in imaging of the central nervous system. First, we focus on improvements in the spatial and temporal profile afforded by optical coherence tomography, fluorescence-guided surgery, and Coherent Anti-Stokes Raman Scattering Microscopy. Next, we highlight advances in the generation and uses of imaging-based atlases and discuss how this will be applied to specific clinical situations. To conclude, we discuss how these and other imaging tools will be combined with neuronavigation techniques to guide surgeons in the operating room. Collectively, this work aims to highlight emerging biomedical imaging strategies that hold potential to be a valuable tool for both clinicians and researchers in the years to come. © 2015 Elsevier B.V. All rights reserved.

Progress in the Correlative Atomic Force Microscopy and Optical Microscopy

PubMed Central

Zhou, Lulu; Cai, Mingjun; Tong, Ti; Wang, Hongda

2017-01-01

Atomic force microscopy (AFM) has evolved from the originally morphological imaging technique to a powerful and multifunctional technique for manipulating and detecting the interactions between molecules at nanometer resolution. However, AFM cannot provide the precise information of synchronized molecular groups and has many shortcomings in the aspects of determining the mechanism of the interactions and the elaborate structure due to the limitations of the technology, itself, such as non-specificity and low imaging speed. To overcome the technical limitations, it is necessary to combine AFM with other complementary techniques, such as fluorescence microscopy. The combination of several complementary techniques in one instrument has increasingly become a vital approach to investigate the details of the interactions among molecules and molecular dynamics. In this review, we reported the principles of AFM and optical microscopy, such as confocal microscopy and single-molecule localization microscopy, and focused on the development and use of correlative AFM and optical microscopy. PMID:28441775

Characterization of the Effects of Hyperbaric Oxygen on the Biochemical and Optical Properties of the Bovine Lens.

PubMed

Lim, Julie C; Vaghefi, Ehsan; Li, Bo; Nye-Wood, Mitchell G; Donaldson, Paul J

2016-04-01

To assess the morphologic, biochemical, and optical properties of bovine lenses treated with hyperbaric oxygen. Lenses were exposed to hyperbaric nitrogen (HBN) or hyperbaric oxygen (HBO) for 5 or 15 hours, lens transparency was assessed using bright field microscopy and lens morphology was visualized using confocal microscopy. Lenses were dissected into the outer cortex, inner cortex, and core, and glutathione (GSH) and malondialdehyde (MDA) measured. Gel electrophoresis and Western blotting were used to detect high molecular weight aggregates (HMW) and glutathione mixed protein disulfides (PSSG). T2-weighted MRI was used to measure lens geometry and map the water/protein ratio to allow gradient refractive index (GRIN) profiles to be calculated. Optical modeling software calculated the change in lens optical power, and an anatomically correct model of the light pathway of the bovine eye was used to determine the effects of HBN and HBO on focal length and overall image quality. Lenses were transparent and lens morphology similar between HBN- and HBO-treated lenses. At 5- and 15-hour HBO exposure, GSH and GSSG were depleted and MDA increased in the core. Glutathione mixed protein disulfides were detected in the outer and inner cortex only with no appearance of HMW. Optical changes were detectable only with 15-hour HBO treatment with a decrease in the refractive index of the core, slightly reduced lens thickness, and an increase in optimal focal length, consistent with a hyperopic shift. This system may serve as a model to study changes that occur with advanced aging rather than nuclear cataract formation per se.

Laser inscription of pseudorandom structures for microphotonic diffuser applications.

PubMed

Alqurashi, Tawfiq; Alhosani, Abdulla; Dauleh, Mahmoud; Yetisen, Ali K; Butt, Haider

2018-04-19

Optical diffusers provide a solution for a variety of applications requiring a Gaussian intensity distribution including imaging systems, biomedical optics, and aerospace. Advances in laser ablation processes have allowed the rapid production of efficient optical diffusers. Here, we demonstrate a novel technique to fabricate high-quality glass optical diffusers with cost-efficiency using a continuous CO2 laser. Surface relief pseudorandom microstructures were patterned on both sides of the glass substrates. A numerical simulation of the temperature distribution showed that the CO2 laser drills a 137 μm hole in the glass for every 2 ms of processing time. FFT simulation was utilized to design predictable optical diffusers. The pseudorandom microstructures were characterized by optical microscopy, Raman spectroscopy, and angle-resolved spectroscopy to assess their chemical properties, optical scattering, transmittance, and polarization response. Increasing laser exposure and the number of diffusing surfaces enhanced the diffusion and homogenized the incident light. The recorded speckle pattern showed high contrast with sharp bright spot free diffusion in the far field view range (250 mm). A model of glass surface peeling was also developed to prevent its occurrence during the fabrication process. The demonstrated method provides an economical approach in fabricating optical glass diffusers in a controlled and predictable manner. The produced optical diffusers have application in fibre optics, LED systems, and spotlights.

Optofluidic Cell Selection from Complex Microbial Communities for Single-Genome Analysis

PubMed Central

Landry, Zachary C.; Giovanonni, Stephen J.; Quake, Stephen R.; Blainey, Paul C.

2013-01-01

Genetic analysis of single cells is emerging as a powerful approach for studies of heterogeneous cell populations. Indeed, the notion of homogeneous cell populations is receding as approaches to resolve genetic and phenotypic variation between single cells are applied throughout the life sciences. A key step in single-cell genomic analysis today is the physical isolation of individual cells from heterogeneous populations, particularly microbial populations, which often exhibit high diversity. Here, we detail the construction and use of instrumentation for optical trapping inside microfluidic devices to select individual cells for analysis by methods including nucleic acid sequencing. This approach has unique advantages for analyses of rare community members, cells with irregular morphologies, small quantity samples, and studies that employ advanced optical microscopy. PMID:24060116

Generation of multiphoton entangled quantum states by means of integrated frequency combs.

PubMed

Reimer, Christian; Kues, Michael; Roztocki, Piotr; Wetzel, Benjamin; Grazioso, Fabio; Little, Brent E; Chu, Sai T; Johnston, Tudor; Bromberg, Yaron; Caspani, Lucia; Moss, David J; Morandotti, Roberto

2016-03-11

Complex optical photon states with entanglement shared among several modes are critical to improving our fundamental understanding of quantum mechanics and have applications for quantum information processing, imaging, and microscopy. We demonstrate that optical integrated Kerr frequency combs can be used to generate several bi- and multiphoton entangled qubits, with direct applications for quantum communication and computation. Our method is compatible with contemporary fiber and quantum memory infrastructures and with chip-scale semiconductor technology, enabling compact, low-cost, and scalable implementations. The exploitation of integrated Kerr frequency combs, with their ability to generate multiple, customizable, and complex quantum states, can provide a scalable, practical, and compact platform for quantum technologies. Copyright © 2016, American Association for the Advancement of Science.

Novel imaging technologies for characterization of microbial extracellular polysaccharides.

PubMed

Lilledahl, Magnus B; Stokke, Bjørn T

2015-01-01

Understanding of biology is underpinned by the ability to observe structures at various length scales. This is so in a historical context and is also valid today. Evolution of novel insight often emerges from technological advancement. Recent developments in imaging technologies that is relevant for characterization of extraceullar microbiological polysaccharides are summarized. Emphasis is on scanning probe and optical based techniques since these tools offers imaging capabilities under aqueous conditions more closely resembling the physiological state than other ultramicroscopy imaging techniques. Following the demonstration of the scanning probe microscopy principle, novel operation modes to increase data capture speed toward video rate, exploitation of several cantilever frequencies, and advancement of utilization of specimen mechanical properties as contrast, also including their mode of operation in liquid, have been developed on this platform. Combined with steps in advancing light microscopy with resolution beyond the far field diffraction limit, non-linear methods, and combinations of the various imaging modalities, the potential ultramicroscopy toolbox available for characterization of exopolysaccharides (EPS) are richer than ever. Examples of application of such ultramicroscopy strategies range from imaging of isolated microbial polysaccharides, structures being observed when they are involved in polyelectrolyte complexes, aspects of their enzymatic degradation, and cell surface localization of secreted polysaccharides. These, and other examples, illustrate that the advancement in imaging technologies relevant for EPS characterization supports characterization of structural aspects.

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.

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

PubMed Central

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

2013-01-01

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

Origins and demonstrations of electrons with orbital angular momentum

PubMed Central

Agrawal, Amit; Ercius, Peter A.; Grillo, Vincenzo; Herzing, Andrew A.; Harvey, Tyler R.; Linck, Martin; Pierce, Jordan S.

2017-01-01

The surprising message of Allen et al. (Allen et al. 1992 Phys. Rev. A 45, 8185 (doi:10.1103/PhysRevA.45.8185)) was that photons could possess orbital angular momentum in free space, which subsequently launched advancements in optical manipulation, microscopy, quantum optics, communications, many more fields. It has recently been shown that this result also applies to quantum mechanical wave functions describing massive particles (matter waves). This article discusses how electron wave functions can be imprinted with quantized phase vortices in analogous ways to twisted light, demonstrating that charged particles with non-zero rest mass can possess orbital angular momentum in free space. With Allen et al. as a bridge, connections are made between this recent work in electron vortex wave functions and much earlier works, extending a 175 year old tradition in matter wave vortices. This article is part of the themed issue ‘Optical orbital angular momentum’. PMID:28069765

Structural characterization on in vitro porcine skin treated by ablative fractional laser using optical coherence tomography

NASA Astrophysics Data System (ADS)

Feng, Kairui; Zhou, Kanheng; Ling, Yuting; O'Mahoney, Paul; Ewan, Eadie; Ibbotson, Sally H.; Li, Chunhui; Huang, Zhihong

2018-02-01

Ablative fractional skin laser is widely applied for various skin conditions, especially for cosmetic repairing and promoting the located drug delivery. Although the influence of laser treatment over the skin has been explored before in means of excision and biopsy with microscopy, these approaches are invasive, only morphological and capable of distorting the skin. In this paper the authors use fresh porcine skin samples irradiated by the lasers, followed by detected by using Optical Coherence Tomography (OCT). This advanced optical technique has the ability to present the high resolution structure image of treated sample. The results shows that laser beams can produce holes left on the surface after the irradiation. The depth of holes can be affected by changes of laser energy while the diameter of holes have no corresponding relation. Plus, OCT, as a valuable imaging technology, is capable of monitoring the clinical therapy procedure and assisting the calibration.

Technologies for imaging neural activity in large volumes

PubMed Central

Ji, Na; Freeman, Jeremy; Smith, Spencer L.

2017-01-01

Neural circuitry has evolved to form distributed networks that act dynamically across large volumes. Collecting data from individual planes, conventional microscopy cannot sample circuitry across large volumes at the temporal resolution relevant to neural circuit function and behaviors. Here, we review emerging technologies for rapid volume imaging of neural circuitry. We focus on two critical challenges: the inertia of optical systems, which limits image speed, and aberrations, which restrict the image volume. Optical sampling time must be long enough to ensure high-fidelity measurements, but optimized sampling strategies and point spread function engineering can facilitate rapid volume imaging of neural activity within this constraint. We also discuss new computational strategies for the processing and analysis of volume imaging data of increasing size and complexity. Together, optical and computational advances are providing a broader view of neural circuit dynamics, and help elucidate how brain regions work in concert to support behavior. PMID:27571194

In situ wavefront correction and its application to micromanipulation

NASA Astrophysics Data System (ADS)

Čižmár, Tomáš; Mazilu, Michael; Dholakia, Kishan

2010-06-01

In any optical system, distortions to a propagating wavefront reduce the spatial coherence of a light field, making it increasingly difficult to obtain the theoretical diffraction-limited spot size. Such aberrations are severely detrimental to optimal performance in imaging, nanosurgery, nanofabrication and micromanipulation, as well as other techniques within modern microscopy. We present a generic method based on complex modulation for true in situ wavefront correction that allows compensation of all aberrations along the entire optical train. The power of the method is demonstrated for the field of micromanipulation, which is very sensitive to wavefront distortions. We present direct trapping with optimally focused laser light carrying power of a fraction of a milliwatt as well as the first trapping through highly turbid and diffusive media. This opens up new perspectives for optical micromanipulation in colloidal and biological physics and may be useful for various forms of advanced imaging.

Micromechanical response of articular cartilage to tensile load measured using nonlinear microscopy.

PubMed

Bell, J S; Christmas, J; Mansfield, J C; Everson, R M; Winlove, C P

2014-06-01

Articular cartilage (AC) is a highly anisotropic biomaterial, and its complex mechanical properties have been a topic of intense investigation for over 60 years. Recent advances in the field of nonlinear optics allow the individual constituents of AC to be imaged in living tissue without the need for exogenous contrast agents. Combining mechanical testing with nonlinear microscopy provides a wealth of information about microscopic responses to load. This work investigates the inhomogeneous distribution of strain in loaded AC by tracking the movement and morphological changes of individual chondrocytes using point pattern matching and Bayesian modeling. This information can be used to inform models of mechanotransduction and pathogenesis, and is readily extendable to various other connective tissues. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Nanotubes, nanobelts, nanowires, and nanorods of silicon carbide from the wheat husks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qadri, S. B.; Rath, B. B.; Gorzkowski, E. P.

2015-09-14

Nanotubes, nanowires, nanobelts, and nanorods of SiC were synthesized from the thermal treatment of wheat husks at temperatures in excess of 1450 °C. From the analysis based on x-ray diffraction, Raman spectroscopy, scanning electron microscopy, and transmission electron microscopy, it has been found that the processed samples of wheat husk consisted of 2H and 3C polytypes of SiC exhibiting the nanostructure shapes. These nanostructures of silicon carbide formed from wheat husks are of technological importance for designing advance composites, applications in biotechnology, and electro-optics. The thermodynamics of the formation of SiC is discussed in terms of the rapid solid state reactionmore » between hydrocarbons and silica on the molecular scale, which is inherently present in the wheat husks.« less

Nanotubes, nanobelts, nanowires, and nanorods of silicon carbide from the wheat husks

NASA Astrophysics Data System (ADS)

Qadri, S. B.; Rath, B. B.; Gorzkowski, E. P.; Feng, J.; Qadri, S. N.; Caldwell, J. D.

2015-09-01

Nanotubes, nanowires, nanobelts, and nanorods of SiC were synthesized from the thermal treatment of wheat husks at temperatures in excess of 1450 °C. From the analysis based on x-ray diffraction, Raman spectroscopy, scanning electron microscopy, and transmission electron microscopy, it has been found that the processed samples of wheat husk consisted of 2H and 3C polytypes of SiC exhibiting the nanostructure shapes. These nanostructures of silicon carbide formed from wheat husks are of technological importance for designing advance composites, applications in biotechnology, and electro-optics. The thermodynamics of the formation of SiC is discussed in terms of the rapid solid state reaction between hydrocarbons and silica on the molecular scale, which is inherently present in the wheat husks.

Adaptive optical fluorescence microscopy.

PubMed

Ji, Na

2017-03-31

The past quarter century has witnessed rapid developments of fluorescence microscopy techniques that enable structural and functional imaging of biological specimens at unprecedented depth and resolution. The performance of these methods in multicellular organisms, however, is degraded by sample-induced optical aberrations. Here I review recent work on incorporating adaptive optics, a technology originally applied in astronomical telescopes to combat atmospheric aberrations, to improve image quality of fluorescence microscopy for biological imaging.

Detection, Mapping, and Quantification of Single Walled Carbon Nanotubes in Histological Specimens with Photoacoustic Microscopy

PubMed Central

Mikos, Antonios G.; Jansen, John A.; Shroyer, Kenneth R.; Wang, Lihong V.; Sitharaman, Balaji

2012-01-01

Aims In the present study, the efficacy of multi-scale photoacoustic microscopy (PAM) was investigated to detect, map, and quantify trace amounts [nanograms (ng) to micrograms (µg)] of SWCNTs in a variety of histological tissue specimens consisting of cancer and benign tissue biopsies (histological specimens from implanted tissue engineering scaffolds). Materials and Methods Optical-resolution (OR) and acoustic-resolution (AR) - Photoacoustic microscopy (PAM) was employed to detect, map and quantify the SWCNTs in a variety of tissue histological specimens and compared with other optical techniques (bright-field optical microscopy, Raman microscopy, near infrared (NIR) fluorescence microscopy). Results Both optical-resolution and acoustic-resolution PAM, allow the detection and quantification of SWCNTs in histological specimens with scalable spatial resolution and depth penetration. The noise-equivalent detection sensitivity to SWCNTs in the specimens was calculated to be as low as ∼7 pg. Image processing analysis further allowed the mapping, distribution, and quantification of the SWCNTs in the histological sections. Conclusions The results demonstrate the potential of PAM as a promising imaging technique to detect, map, and quantify SWCNTs in histological specimens, and could complement the capabilities of current optical and electron microscopy techniques in the analysis of histological specimens containing SWCNTs. PMID:22496892

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

Maximizing the Biochemical Resolving Power of Fluorescence Microscopy

PubMed Central

Esposito, Alessandro; Popleteeva, Marina; Venkitaraman, Ashok R.

2013-01-01

Most recent advances in fluorescence microscopy have focused on achieving spatial resolutions below the diffraction limit. However, the inherent capability of fluorescence microscopy to non-invasively resolve different biochemical or physical environments in biological samples has not yet been formally described, because an adequate and general theoretical framework is lacking. Here, we develop a mathematical characterization of the biochemical resolution in fluorescence detection with Fisher information analysis. To improve the precision and the resolution of quantitative imaging methods, we demonstrate strategies for the optimization of fluorescence lifetime, fluorescence anisotropy and hyperspectral detection, as well as different multi-dimensional techniques. We describe optimized imaging protocols, provide optimization algorithms and describe precision and resolving power in biochemical imaging thanks to the analysis of the general properties of Fisher information in fluorescence detection. These strategies enable the optimal use of the information content available within the limited photon-budget typically available in fluorescence microscopy. This theoretical foundation leads to a generalized strategy for the optimization of multi-dimensional optical detection, and demonstrates how the parallel detection of all properties of fluorescence can maximize the biochemical resolving power of fluorescence microscopy, an approach we term Hyper Dimensional Imaging Microscopy (HDIM). Our work provides a theoretical framework for the description of the biochemical resolution in fluorescence microscopy, irrespective of spatial resolution, and for the development of a new class of microscopes that exploit multi-parametric detection systems. PMID:24204821

Strong Electro‐Optic Effect and Spontaneous Domain Formation in Self‐Assembled Peptide Structures

PubMed Central

Lafargue, Clément; Handelman, Amir; Shimon, Linda J. W.; Rosenman, Gil; Zyss, Joseph

2017-01-01

Short peptides made from repeating units of phenylalanine self‐assemble into a remarkable variety of micro‐ and nanostructures including tubes, tapes, spheres, and fibrils. These bio‐organic structures are found to possess striking mechanical, electrical, and optical properties, which are rarely seen in organic materials, and are therefore shown useful for diverse applications including regenerative medicine, targeted drug delivery, and biocompatible fluorescent probes. Consequently, finding new optical properties in these materials can significantly advance their practical use, for example, by allowing new ways to visualize, manipulate, and utilize them in new, in vivo, sensing applications. Here, by leveraging a unique electro‐optic phase microscopy technique, combined with traditional structural analysis, it is measured in di‐ and triphenylalanine peptide structures a surprisingly large electro‐optic response of the same order as the best performing inorganic crystals. In addition, spontaneous domain formation is observed in triphenylalanine tapes, and the origin of their electro‐optic activity is unveiled to be related to a porous triclinic structure, with extensive antiparallel beta‐sheet arrangement. The strong electro‐optic response of these porous peptide structures with the capability of hosting guest molecules opens the door to create new biocompatible, environmental friendly functional materials for electro‐optic applications, including biomedical imaging, sensing, and optical manipulation. PMID:28932664

Characterization of new drug delivery nanosystems using atomic force microscopy

NASA Astrophysics Data System (ADS)

Spyratou, Ellas; Mourelatou, Elena A.; Demetzos, C.; Makropoulou, Mersini; Serafetinides, A. A.

2015-01-01

Liposomes are the most attractive lipid vesicles for targeted drug delivery in nanomedicine, behaving also as cell models in biophotonics research. The characterization of the micro-mechanical properties of drug carriers is an important issue and many analytical techniques are employed, as, for example, optical tweezers and atomic force microscopy. In this work, polyol hyperbranched polymers (HBPs) have been employed along with liposomes for the preparation of new chimeric advanced drug delivery nanosystems (Chi-aDDnSs). Aliphatic polyester HBPs with three different pseudogenerations G2, G3 and G4 with 16, 32, and 64 peripheral hydroxyl groups, respectively, have been incorporated in liposomal formulation. The atomic force microscopy (AFM) technique was used for the comparative study of the morphology and the mechanical properties of Chi-aDDnSs and conventional DDnS. The effects of both the HBPs architecture and the polyesters pseudogeneration number in the stability and the stiffness of chi-aDDnSs were examined. From the force-distance curves of AFM spectroscopy, the Young's modulus was calculated.

High throughput, detailed, cell-specific neuroanatomy of dendritic spines using microinjection and confocal microscopy

PubMed Central

Dumitriu, Dani; Rodriguez, Alfredo; Morrison, John H.

2012-01-01

Morphological features such as size, shape and density of dendritic spines have been shown to reflect important synaptic functional attributes and potential for plasticity. Here we describe in detail a protocol for obtaining detailed morphometric analysis of spines using microinjection of fluorescent dyes, high resolution confocal microscopy, deconvolution and image analysis using NeuronStudio. Recent technical advancements include better preservation of tissue resulting in prolonged ability to microinject, and algorithmic improvements that compensate for the residual Z-smear inherent in all optical imaging. Confocal imaging parameters were probed systematically for the identification of both optimal resolution as well as highest efficiency. When combined, our methods yield size and density measurements comparable to serial section transmission electron microscopy in a fraction of the time. An experiment containing 3 experimental groups with 8 subjects in each can take as little as one month if optimized for speed, or approximately 4 to 5 months if the highest resolution and morphometric detail is sought. PMID:21886104

In Situ Identification of Nanoparticle Structural Information Using Optical Microscopy.

PubMed

Culver, Kayla S B; Liu, Tingting; Hryn, Alexander J; Fang, Ning; Odom, Teri W

2018-05-11

Diffraction-limited optical microscopy lacks the resolution to characterize directly nanoscale features of single nanoparticles. This paper describes how surprisingly rich structural features of small gold nanostars can be identified using differential interference contrast (DIC) microscopy. First, we established a library of structure-property relationships between nanoparticle shape and DIC optical image and then validated the correlation with electrodynamic simulations and electron microscopy. We found that DIC image patterns of single nanostars could be differentiated between 2D and 3D geometries. Also, DIC images could elucidate the symmetry properties and orientation of nanoparticles. Finally, we demonstrated how this wide-field optical technique can be used for in situ characterization of single nanoparticles rotating at a glass-water interface.

Stochastic optical reconstruction microscopy-based relative localization analysis (STORM-RLA) for quantitative nanoscale assessment of spatial protein organization.

PubMed

Veeraraghavan, Rengasayee; Gourdie, Robert G

2016-11-07

The spatial association between proteins is crucial to understanding how they function in biological systems. Colocalization analysis of fluorescence microscopy images is widely used to assess this. However, colocalization analysis performed on two-dimensional images with diffraction-limited resolution merely indicates that the proteins are within 200-300 nm of each other in the xy-plane and within 500-700 nm of each other along the z-axis. Here we demonstrate a novel three-dimensional quantitative analysis applicable to single-molecule positional data: stochastic optical reconstruction microscopy-based relative localization analysis (STORM-RLA). This method offers significant advantages: 1) STORM imaging affords 20-nm resolution in the xy-plane and <50 nm along the z-axis; 2) STORM-RLA provides a quantitative assessment of the frequency and degree of overlap between clusters of colabeled proteins; and 3) STORM-RLA also calculates the precise distances between both overlapping and nonoverlapping clusters in three dimensions. Thus STORM-RLA represents a significant advance in the high-throughput quantitative assessment of the spatial organization of proteins. © 2016 Veeraraghavan and Gourdie. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

In vivo measurements of cutaneous melanin across spatial scales: using multiphoton microscopy and spatial frequency domain spectroscopy

PubMed Central

Saager, Rolf B.; Balu, Mihaela; Crosignani, Viera; Sharif, Ata; Durkin, Anthony J.; Kelly, Kristen M.; Tromberg, Bruce J.

2015-01-01

Abstract. The combined use of nonlinear optical microscopy and broadband reflectance techniques to assess melanin concentration and distribution thickness in vivo over the full range of Fitzpatrick skin types is presented. Twelve patients were measured using multiphoton microscopy (MPM) and spatial frequency domain spectroscopy (SFDS) on both dorsal forearm and volar arm, which are generally sun-exposed and non-sun-exposed areas, respectively. Both MPM and SFDS measured melanin volume fractions between ∼5% (skin type I non-sun-exposed) and 20% (skin type VI sun exposed). MPM measured epidermal (anatomical) thickness values ∼30–65  μm, while SFDS measured melanin distribution thickness based on diffuse optical path length. There was a strong correlation between melanin concentration and melanin distribution (epidermal) thickness measurements obtained using the two techniques. While SFDS does not have the ability to match the spatial resolution of MPM, this study demonstrates that melanin content as quantified using SFDS is linearly correlated with epidermal melanin as measured using MPM (R2=0.8895). SFDS melanin distribution thickness is correlated to MPM values (R2=0.8131). These techniques can be used individually and/or in combination to advance our understanding and guide therapies for pigmentation-related conditions as well as light-based treatments across a full range of skin types. PMID:26065839

Hybrid microscopy of human carotid atheroma by means of optical-resolution optoacoustic and non-linear optical microscopy

NASA Astrophysics Data System (ADS)

Seeger, Markus; Karlas, Angelos; Soliman, Dominik; Pelisek, Jaroslav; Ntziachristos, Vasilis

2017-03-01

Carotid atheromatosis is causally related to stroke, a leading cause of disability and death. We present the analysis of a human carotid atheroma using a novel hybrid microscopy system that combines optical-resolution optoacoustic (photoacoustic) microscopy and several non-linear optical microscopy modalities (second and third harmonic generation, as well as, two-photon excitation fluorescence) to achieve a multimodal examination of the extracted tissue within the same imaging framework. Our system enables the label-free investigation of atheromatous human carotid tissue with a resolution of about 1 μm and allows for the congruent interrogation of plaque morphology and clinically relevant constituents such as red blood cells, collagen, and elastin. Our data reveal mutual interactions between blood embeddings and connective tissue within the atheroma, offering comprehensive insights into its stage of evolution and severity, and potentially facilitating the further development of diagnostic tools, as well as treatment strategies.

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. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

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 spontaneous Raman spectroscopy. We used synthesized highly-deuterated cholesterol to track its compartmentalization in adrenal cells, revealing heterogeneous lipid droplet content. These examples illustrate the potential of label-free nonlinear optical microscopy for unveiling complex physiological processes by direct visualization of lipids. Detailed image analysis and combined microscopy modalities will continue to reveal and quantify fundamental biology that will support the advance of biomedicine.

A multimodal imaging platform with integrated simultaneous photoacoustic microscopy, optical coherence tomography, optical Doppler tomography and fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dadkhah, Arash; Zhou, Jun; Yeasmin, Nusrat; Jiao, Shuliang

2018-02-01

Various optical imaging modalities with different optical contrast mechanisms have been developed over the past years. Although most of these imaging techniques are being used in many biomedical applications and researches, integration of these techniques will allow researchers to reach the full potential of these technologies. Nevertheless, combining different imaging techniques is always challenging due to the difference in optical and hardware requirements for different imaging systems. Here, we developed a multimodal optical imaging system with the capability of providing comprehensive structural, functional and molecular information of living tissue in micrometer scale. This imaging system integrates photoacoustic microscopy (PAM), optical coherence tomography (OCT), optical Doppler tomography (ODT) and fluorescence microscopy in one platform. Optical-resolution PAM (OR-PAM) provides absorption-based imaging of biological tissues. Spectral domain OCT is able to provide structural information based on the scattering property of biological sample with no need for exogenous contrast agents. In addition, ODT is a functional extension of OCT with the capability of measurement and visualization of blood flow based on the Doppler effect. Fluorescence microscopy allows to reveal molecular information of biological tissue using autofluoresce or exogenous fluorophores. In-vivo as well as ex-vivo imaging studies demonstrated the capability of our multimodal imaging system to provide comprehensive microscopic information on biological tissues. Integrating all the aforementioned imaging modalities for simultaneous multimodal imaging has promising potential for preclinical research and clinical practice in the near future.

High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events

PubMed Central

Suzuki, Yuki; Sakai, Nobuaki; Yoshida, Aiko; Uekusa, Yoshitsugu; Yagi, Akira; Imaoka, Yuka; Ito, Shuichi; Karaki, Koichi; Takeyasu, Kunio

2013-01-01

A hybrid atomic force microscopy (AFM)-optical fluorescence microscopy is a powerful tool for investigating cellular morphologies and events. However, the slow data acquisition rates of the conventional AFM unit of the hybrid system limit the visualization of structural changes during cellular events. Therefore, high-speed AFM units equipped with an optical/fluorescence detection device have been a long-standing wish. Here we describe the implementation of high-speed AFM coupled with an optical fluorescence microscope. This was accomplished by developing a tip-scanning system, instead of a sample-scanning system, which operates on an inverted optical microscope. This novel device enabled the acquisition of high-speed AFM images of morphological changes in individual cells. Using this instrument, we conducted structural studies of living HeLa and 3T3 fibroblast cell surfaces. The improved time resolution allowed us to image dynamic cellular events. PMID:23823461

High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events.

PubMed

Suzuki, Yuki; Sakai, Nobuaki; Yoshida, Aiko; Uekusa, Yoshitsugu; Yagi, Akira; Imaoka, Yuka; Ito, Shuichi; Karaki, Koichi; Takeyasu, Kunio

2013-01-01

A hybrid atomic force microscopy (AFM)-optical fluorescence microscopy is a powerful tool for investigating cellular morphologies and events. However, the slow data acquisition rates of the conventional AFM unit of the hybrid system limit the visualization of structural changes during cellular events. Therefore, high-speed AFM units equipped with an optical/fluorescence detection device have been a long-standing wish. Here we describe the implementation of high-speed AFM coupled with an optical fluorescence microscope. This was accomplished by developing a tip-scanning system, instead of a sample-scanning system, which operates on an inverted optical microscope. This novel device enabled the acquisition of high-speed AFM images of morphological changes in individual cells. Using this instrument, we conducted structural studies of living HeLa and 3T3 fibroblast cell surfaces. The improved time resolution allowed us to image dynamic cellular events.

Review of combined isotopic and optical nanoscopy

PubMed Central

Richter, Katharina N.; Rizzoli, Silvio O.; Jähne, Sebastian; Vogts, Angela; Lovric, Jelena

2017-01-01

Abstract. Investigating the detailed substructure of the cell is beyond the ability of conventional optical microscopy. Electron microscopy, therefore, has been the only option for such studies for several decades. The recent implementation of several super-resolution optical microscopy techniques has rendered the investigation of cellular substructure easier and more efficient. Nevertheless, optical microscopy only provides an image of the present structure of the cell, without any information on its long-temporal changes. These can be investigated by combining super-resolution optics with a nonoptical imaging technique, nanoscale secondary ion mass spectrometry, which investigates the isotopic composition of the samples. The resulting technique, combined isotopic and optical nanoscopy, enables the investigation of both the structure and the “history” of the cellular elements. The age and the turnover of cellular organelles can be read by isotopic imaging, while the structure can be analyzed by optical (fluorescence) approaches. We present these technologies, and we discuss their implementation for the study of biological samples. We conclude that, albeit complex, this type of technology is reliable enough for mass application to cell biology. PMID:28466025

Coherent optical adaptive technique improves the spatial resolution of STED microscopy in thick samples

PubMed Central

Yan, Wei; Yang, Yanlong; Tan, Yu; Chen, Xun; Li, Yang; Qu, Junle; Ye, Tong

2018-01-01

Stimulated emission depletion microscopy (STED) is one of far-field optical microscopy techniques that can provide sub-diffraction spatial resolution. The spatial resolution of the STED microscopy is determined by the specially engineered beam profile of the depletion beam and its power. However, the beam profile of the depletion beam may be distorted due to aberrations of optical systems and inhomogeneity of specimens’ optical properties, resulting in a compromised spatial resolution. The situation gets deteriorated when thick samples are imaged. In the worst case, the sever distortion of the depletion beam profile may cause complete loss of the super resolution effect no matter how much depletion power is applied to specimens. Previously several adaptive optics approaches have been explored to compensate aberrations of systems and specimens. However, it is hard to correct the complicated high-order optical aberrations of specimens. In this report, we demonstrate that the complicated distorted wavefront from a thick phantom sample can be measured by using the coherent optical adaptive technique (COAT). The full correction can effectively maintain and improve the spatial resolution in imaging thick samples. PMID:29400356

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. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy.

Enabling the detection of UV signal in multimodal nonlinear microscopy with catalogue lens components.

PubMed

Vogel, Martin; Wingert, Axel; Fink, Rainer H A; Hagl, Christian; Ganikhanov, Feruz; Pfeffer, Christian P

2015-10-01

Using an optical system made from fused silica catalogue optical components, third-order nonlinear microscopy has been enabled on conventional Ti:sapphire laser-based multiphoton microscopy setups. The optical system is designed using two lens groups with straightforward adaptation to other microscope stands when one of the lens groups is exchanged. Within the theoretical design, the optical system collects and transmits light with wavelengths between the near ultraviolet and the near infrared from an object field of at least 1 mm in diameter within a resulting numerical aperture of up to 0.56. The numerical aperture can be controlled with a variable aperture stop between the two lens groups of the condenser. We demonstrate this new detection capability in third harmonic generation imaging experiments at the harmonic wavelength of ∼300 nm and in multimodal nonlinear optical imaging experiments using third-order sum frequency generation and coherent anti-Stokes Raman scattering microscopy so that the wavelengths of the detected signals range from ∼300 nm to ∼660 nm. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

An ultrafast electron microscope gun driven by two-photon photoemission from a nanotip cathode

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bormann, Reiner; Strauch, Stefanie; Schäfer, Sascha, E-mail: schaefer@ph4.physik.uni-goettingen.de

We experimentally and numerically investigate the performance of an advanced ultrafast electron source, based on two-photon photoemission from a tungsten needle cathode incorporated in an electron microscope gun geometry. Emission properties are characterized as a function of the electrostatic gun settings, and operating conditions leading to laser-triggered electron beams of very low emittance (below 20 nm mrad) are identified. The results highlight the excellent suitability of optically driven nano-cathodes for the further development of ultrafast transmission electron microscopy.

Increased metabolic activity detected by FLIM in human breast cancer cells with desmoplastic reaction: a pilot study

NASA Astrophysics Data System (ADS)

Natal, Rodrigo de Andrade; Pelegati, Vitor B.; Bondarik, Caroline; Mendonça, Guilherme R.; Derchain, Sophie F.; Lima, Carmen P.; Cesar, Carlos L.; Sarian, Luís. O.; Vassallo, José

2015-07-01

Introduction: In breast cancer (BC), desmoplastic reaction, assembled primarily by fibroblasts, is associated with unfavorable prognosis, but the reason of this fact remains still unclear. In this context, nonlinear optics microscopy, including Fluorescence Lifetime Imaging Microscopy (FLIM), has provided advancement in cellular metabolism research. In this paper, our purpose is to differentiate BC cells metabolism with or without contact to desmoplastic reaction. Formalin fixed, paraffin embedded samples were used at different points of hematoxylin stained sections. Methodology: Sections from 14 patients with invasive ductal breast carcinoma were analyzed with FLIM methodology to NAD(P)H and FAD fluorescence lifetime on a Confocal Upright LSM780 NLO device (Carl Zeiss AG, Germany). Quantification of the fluorescence lifetime and fluorescence intensity was evaluated by SPC Image software (Becker &Hickl) and ImageJ (NIH), respectively. Optical redox ratio was calculated by dividing the FAD fluorescence intensity by NAD(P)H fluorescence intensity. Data value for FLIM measurements and fluorescence intensities were calculated using Wilcoxon test; p< 0.05 was considered significant. Results: BC cells in contact with desmoplastic reaction presented a significantly lower NAD(P)H and FAD fluorescence lifetime. Furthermore, optical redox ratio was also lower in these tumor cells. Conclusion: Our results suggest that contact of BC cells with desmoplastic reaction increase their metabolic activity, which might explain the adverse prognosis of cases associated with higher peritumoral desmoplastic reaction.

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.

Advanced Test Reactor National Scientific User Facility (ATR NSUF) Monthly Report December 2014

DOE Office of Scientific and Technical Information (OSTI.GOV)

Renae Soelberg

2014-12-01

• PNNL has completed sectioning of the U.C. Berkeley hydride fuel rodlet 1 (highest burn-up) and is currently polishing samples in preparation for optical metallography. • A disk was successfully sectioned from rodlet 1 at the location of the internal thermocouple tip as desired. The transition from annular pellet to solid pellet is verified by the eutectic-filled inner cavity located on the back face of this disk (top left) and the solid front face (bottom left). Preliminary low-resolution images indicate interesting sample characteristics in the eutectic surrounding the rodlet at the location of the outer thermocouple tip (right). This samplemore » has been potted and is currently being polished for high-resolution optical microscopy and subsequent SEM analysis. (See images.)« less

From Airy to Abbe: quantifying the effects of wide-angle focusing for scalar spherical waves

NASA Astrophysics Data System (ADS)

Calm, Yitzi M.; Merlo, Juan M.; Burns, Michael J.; Naughton, Michael J.

2017-10-01

Recent advances in optical microscopy have enabled imaging with spatial resolution beyond the diffraction limit. This limit is sometimes taken as one of several different criteria according to different conventions, including Rayleigh’s 0.61λ /NA, Abbe’s 0.5λ /NA, and Sparrow’s 0.47λ /NA. In this paper, we perform a parametric study, numerically integrating the scalar Kirchhoff diffraction integrals, and we propose new functional forms for the resolution limits derived from scalar focusing. The new expressions remain accurate under wide angle focusing, up to 90^\\circ . Our results could materially impact the design of high intensity focused ultrasound systems, and can be used as a qualitative guideline for the design of a particular type of planar optical element: the flat lens metasurface.

Large-angle illumination STEM: Toward three-dimensional atom-by-atom imaging

DOE PAGES

Ishikawa, Ryo; Lupini, Andrew R.; Hinuma, Yoyo; ...

2014-11-26

To completely understand and control materials and their properties, it is of critical importance to determine their atomic structures in all three dimensions. Recent revolutionary advances in electron optics – the inventions of geometric and chromatic aberration correctors as well as electron source monochromators – have provided fertile ground for performing optical depth sectioning at atomic-scale dimensions. In this study we theoretically demonstrate the imaging of top/sub-surface atomic structures and identify the depth of single dopants, single vacancies and the other point defects within materials by large-angle illumination scanning transmission electron microscopy (LAI-STEM). The proposed method also allows us tomore » measure specimen properties such as thickness or three-dimensional surface morphology using observations from a single crystallographic orientation.« less

Improved wavefront correction for coherent image restoration.

PubMed

Zelenka, Claudius; Koch, Reinhard

2017-08-07

Coherent imaging has a wide range of applications in, for example, microscopy, astronomy, and radar imaging. Particularly interesting is the field of microscopy, where the optical quality of the lens is the main limiting factor. In this article, novel algorithms for the restoration of blurred images in a system with known optical aberrations are presented. Physically motivated by the scalar diffraction theory, the new algorithms are based on Haugazeau POCS and FISTA, and are faster and more robust than methods presented earlier. With the new approach the level of restoration quality on real images is very high, thereby blurring and ringing caused by defocus can be effectively removed. In classical microscopy, lenses with very low aberration must be used, which puts a practical limit on their size and numerical aperture. A coherent microscope using the novel restoration method overcomes this limitation. In contrast to incoherent microscopy, severe optical aberrations including defocus can be removed, hence the requirements on the quality of the optics are lower. This can be exploited for an essential price reduction of the optical system. It can be also used to achieve higher resolution than in classical microscopy, using lenses with high numerical aperture and high aberration. All this makes the coherent microscopy superior to the traditional incoherent in suited applications.

Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed, and resolution.

PubMed

Coates, Colin G; Denvir, Donal J; McHale, Noel G; Thornbury, Keith D; Hollywood, Mark A

2004-01-01

The back-illuminated electron multiplying charge-coupled device (EMCCD) camera is having a profound influence on the field of low-light dynamic cellular microscopy, combining highest possible photon collection efficiency with the ability to virtually eliminate the readout noise detection limit. We report here the use of this camera, in 512 x 512 frame-transfer chip format at 10-MHz pixel readout speed, in optimizing a demanding ultra-low-light intracellular calcium flux microscopy setup. The arrangement employed includes a spinning confocal Nipkow disk, which, while facilitating the need to both generate images at very rapid frame rates and minimize background photons, yields very weak signals. The challenge for the camera lies not just in detecting as many of these scarce photons as possible, but also in operating at a frame rate that meets the temporal resolution requirements of many low-light microscopy approaches, a particular demand of smooth muscle calcium flux microscopy. Results presented illustrate both the significant sensitivity improvement offered by this technology over the previous standard in ultra-low-light CCD detection, the GenIII+intensified charge-coupled device (ICCD), and also portray the advanced temporal and spatial resolution capabilities of the EMCCD. Copyright 2004 Society of Photo-Optical Instrumentation Engineers.

Application of carbon nanotubes to topographical resolution enhancement of tapered fiber scanning near field optical microscopy probes

NASA Astrophysics Data System (ADS)

Huntington, S. T.; Jarvis, S. P.

2003-05-01

Scanning near field optical microscopy (SNOM) probes are typically tapered optical fibers with metallic coatings. The tip diameters are generally in excess of 300 nm and thus provide poor topographical resolution. Here we report on the attachment multiwalled carbon nanotubes to the probes in order to substantially enhance the topographical resolution, without adversely affecting the optical resolution.

Time to Stop Telling Biophysics Students that Light Is Primarily a Wave.

PubMed

Nelson, Philip C

2018-02-27

Standard pedagogy introduces optics as though it were a consequence of Maxwell's equations and only grudgingly admits, usually in a rushed aside, that light has a particulate character that can somehow be reconciled with the wave picture. Recent revolutionary advances in optical imaging, however, make this approach more and more unhelpful: How are we to describe two-photon imaging, FRET, localization microscopy, and a host of related techniques to students who think of light primarily as a wave? I was surprised to find that everything I wanted my biophysics students to know about light, including image formation, x-ray diffraction, and even Bessel beams, could be expressed as well (or better) from the quantum viewpoint pioneered by Richard Feynman. Even my undergraduate students grasp this viewpoint as well as (or better than) the traditional one, and by mid-semester they are already well positioned to integrate the latest advances into their understanding. Moreover, I have found that this approach clarifies my own understanding of new techniques. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Three-dimensional optical-transfer-function analysis of fiber-optical two-photon fluorescence microscopy.

PubMed

Gu, Min; Bird, Damian

2003-05-01

The three-dimensional optical transfer function is derived for analyzing the imaging performance in fiber-optical two-photon fluorescence microscopy. Two types of fiber-optical geometry are considered: The first involves a single-mode fiber for delivering a laser beam for illumination, and the second is based on the use of a single-mode fiber coupler for both illumination delivery and signal collection. It is found that in the former case the transverse and axial cutoff spatial frequencies of the three-dimensional optical transfer function are the same as those in conventional two-photon fluorescence microscopy without the use of a pinhole.However, the transverse and axial cutoff spatial frequencies in the latter case are 1.7 times as large as those in the former case. Accordingly, this feature leads to an enhanced optical sectioning effect when a fiber coupler is used, which is consistent with our recent experimental observation.

Molecular expressions: exploring the world of optics and microscopy. http://microscopy.fsu.edu.

PubMed

Eliceiri, Kevin W

2004-08-01

Our knowledge of the structure, dynamics and physiology of a cell has increased significantly in the last ten years through the emergence of new optical imaging modalities such as optical sectioning microscopy, computer- enhanced video microscopy and laser-scanning microscopy. These techniques together with the use of genetically engineered fluorophores have helped scientists visualize the 3-dimensional dynamic processes of living cells. However as powerful as these imaging tools are, they can often be difficult to understand and fully utilize. Below I will discuss my favorite website: The Molecular Expressions Web Site that endeavors to present the power of microscopy to its visitors. The Molecular Expressions group does a remarkable job of not only clearly presenting the principles behind these techniques in a manner approachable by lay and scientific audiences alike but also provides representative data from each as well.

Imaging slit-coupled surface plasmon polaritons using conventional optical microscopy.

PubMed

Mehfuz, R; Chowdhury, F A; Chau, K J

2012-05-07

We develop a technique that now enables surface plasmon polaritons (SPPs) coupled by nano-patterned slits in a metal film to be detected using conventional optical microscopy with standard objective lenses. The crux of this method is an ultra-thin polymer layer on the metal surface, whose thickness can be varied over a nanoscale range to enable controllable tuning of the SPP momentum. At an optimal layer thickness for which the SPP momentum matches the momentum of light emerging from the slit, the SPP coupling efficiency is enhanced about six times relative to that without the layer. The enhanced efficiency results in distinctive and bright plasmonic signatures near the slit visible by naked eye under an optical microscope. We demonstrate how this capability can be used for parallel measurement through a simple experiment in which the SPP propagation distance is extracted from a single microscope image of an illuminated array of nano-patterned slits on a metal surface. We also use optical microscopy to image the focal region of a plasmonic lens and obtain results consistent with a previously-reported results using near-field optical microscopy. Measurement of SPPs near a nano-slit using conventional and widely-available optical microscopy is an important step towards making nano-plasmonic device technology highly accessible and easy-to-use.

SIL-STED microscopy technique enhancing super-resolution of fluorescence microscopy

NASA Astrophysics Data System (ADS)

Park, No-Cheol; Lim, Geon; Lee, Won-sup; Moon, Hyungbae; Choi, Guk-Jong; Park, Young-Pil

2017-08-01

We have characterized a new type STED microscope which combines a high numerical aperture (NA) optical head with a solid immersion lens (SIL), and we call it as SIL-STED microscope. The advantage of a SIL-STED microscope is that its high NA of the SIL makes it superior to a general STED microscope in lateral resolution, thus overcoming the optical diffraction limit at the macromolecular level and enabling advanced super-resolution imaging of cell surface or cell membrane structure and function Do. This study presents the first implementation of higher NA illumination in a STED microscope limiting the fluorescence lateral resolution to about 40 nm. The refractive index of the SIL which is made of material KTaO3 is about 2.23 and 2.20 at a wavelength of 633 nm and 780 nm which are used for excitation and depletion in STED imaging, respectively. Based on the vector diffraction theory, the electric field focused by the SILSTED microscope is numerically calculated so that the numerical results of the point dispersion function of the microscope and the expected resolution could be analyzed. For further investigation, fluorescence imaging of nano size fluorescent beads is fulfilled to show improved performance of the technique.

Visualization of neuritic plaques in Alzheimer’s disease by polarization-sensitive optical coherence microscopy

NASA Astrophysics Data System (ADS)

Baumann, Bernhard; Woehrer, Adelheid; Ricken, Gerda; Augustin, Marco; Mitter, Christian; Pircher, Michael; Kovacs, Gabor G.; Hitzenberger, Christoph K.

2017-03-01

One major hallmark of Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA) is the deposition of extracellular senile plaques and vessel wall deposits composed of amyloid-beta (Aβ). In AD, degeneration of neurons is preceded by the formation of Aβ plaques, which show different morphological forms. Most of them are birefringent owing to the parallel arrangement of amyloid fibrils. Here, we present polarization sensitive optical coherence microscopy (PS-OCM) for imaging mature neuritic Aβ plaques based on their birefringent properties. Formalin-fixed, post-mortem brain samples of advanced stage AD patients were investigated. In several cortical brain regions, neuritic Aβ plaques were successfully visualized in tomographic and three-dimensional (3D) images. Cortical grey matter appeared polarization preserving, whereas neuritic plaques caused increased phase retardation. Consistent with the results from PS-OCM imaging, the 3D structure of senile Aβ plaques was computationally modelled for different illumination settings and plaque sizes. Furthermore, the birefringent properties of cortical and meningeal vessel walls in CAA were investigated in selected samples. Significantly increased birefringence was found in smaller vessels. Overall, these results provide evidence that PS-OCM is able to assess amyloidosis based on intrinsic birefringent properties.

Core-shell designs of photoluminescent nanodiamonds with porous silica coatings for bioimaging and drug delivery II: application.

PubMed

Prabhakar, Neeraj; Näreoja, Tuomas; von Haartman, Eva; Karaman, Didem Şen; Jiang, Hua; Koho, Sami; Dolenko, Tatiana A; Hänninen, Pekka E; Vlasov, Denis I; Ralchenko, Victor G; Hosomi, Satoru; Vlasov, Igor I; Sahlgren, Cecilia; Rosenholm, Jessica M

2013-05-07

Recent advances within materials science and its interdisciplinary applications in biomedicine have emphasized the potential of using a single multifunctional composite material for concurrent drug delivery and biomedical imaging. Here we present a novel composite material consisting of a photoluminescent nanodiamond (ND) core with a porous silica (SiO2) shell. This novel multifunctional probe serves as an alternative nanomaterial to address the existing problems with delivery and subsequent tracing of the particles. Whereas the unique optical properties of ND allows for long-term live cell imaging and tracking of cellular processes, mesoporous silica nanoparticles (MSNs) have proven to be efficient drug carriers. The advantages of both ND and MSNs were hereby integrated in the new composite material, ND@MSN. The optical properties provided by the ND core rendered the nanocomposite suitable for microscopy imaging in fluorescence and reflectance mode, as well as super-resolution microscopy as a STED label; whereas the porous silica coating provided efficient intracellular delivery capacity, especially in surface-functionalized form. This study serves as a demonstration how this novel nanomaterial can be exploited for both bioimaging and drug delivery for future theranostic applications.

Optical coherence tomography - principles and applications

NASA Astrophysics Data System (ADS)

Fercher, A. F.; Drexler, W.; Hitzenberger, C. K.; Lasser, T.

2003-02-01

There have been three basic approaches to optical tomography since the early 1980s: diffraction tomography, diffuse optical tomography and optical coherence tomography (OCT). Optical techniques are of particular importance in the medical field, because these techniques promise to be safe and cheap and, in addition, offer a therapeutic potential. Advances in OCT technology have made it possible to apply OCT in a wide variety of applications but medical applications are still dominating. Specific advantages of OCT are its high depth and transversal resolution, the fact, that its depth resolution is decoupled from transverse resolution, high probing depth in scattering media, contact-free and non-invasive operation, and the possibility to create various function dependent image contrasting methods. This report presents the principles of OCT and the state of important OCT applications. OCT synthesises cross-sectional images from a series of laterally adjacent depth-scans. At present OCT is used in three different fields of optical imaging, in macroscopic imaging of structures which can be seen by the naked eye or using weak magnifications, in microscopic imaging using magnifications up to the classical limit of microscopic resolution and in endoscopic imaging, using low and medium magnification. First, OCT techniques, like the reflectometry technique and the dual beam technique were based on time-domain low coherence interferometry depth-scans. Later, Fourier-domain techniques have been developed and led to new imaging schemes. Recently developed parallel OCT schemes eliminate the need for lateral scanning and, therefore, dramatically increase the imaging rate. These schemes use CCD cameras and CMOS detector arrays as photodetectors. Video-rate three-dimensional OCT pictures have been obtained. Modifying interference microscopy techniques has led to high-resolution optical coherence microscopy that achieved sub-micrometre resolution. This report is concluded with a short presentation of important OCT applications. Ophthalmology is, due to the transparent ocular structures, still the main field of OCT application. The first commercial instrument too has been introduced for ophthalmic diagnostics (Carl Zeiss Meditec AG). Advances in using near-infrared light, however, opened the path for OCT imaging in strongly scattering tissues. Today, optical in vivo biopsy is one of the most challenging fields of OCT application. High resolution, high penetration depth, and its potential for functional imaging attribute to OCT an optical biopsy quality, which can be used to assess tissue and cell function and morphology in situ. OCT can already clarify the relevant architectural tissue morphology. For many diseases, however, including cancer in its early stages, higher resolution is necessary. New broad-bandwidth light sources, like photonic crystal fibres and superfluorescent fibre sources, and new contrasting techniques, give access to new sample properties and unmatched sensitivity and resolution.

Aberrations and adaptive optics in super-resolution microscopy

PubMed Central

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

2015-01-01

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. PMID:26124194

Light sheet theta microscopy for rapid high-resolution imaging of large biological samples.

PubMed

Migliori, Bianca; Datta, Malika S; Dupre, Christophe; Apak, Mehmet C; Asano, Shoh; Gao, Ruixuan; Boyden, Edward S; Hermanson, Ola; Yuste, Rafael; Tomer, Raju

2018-05-29

Advances in tissue clearing and molecular labeling methods are enabling unprecedented optical access to large intact biological systems. These developments fuel the need for high-speed microscopy approaches to image large samples quantitatively and at high resolution. While light sheet microscopy (LSM), with its high planar imaging speed and low photo-bleaching, can be effective, scaling up to larger imaging volumes has been hindered by the use of orthogonal light sheet illumination. To address this fundamental limitation, we have developed light sheet theta microscopy (LSTM), which uniformly illuminates samples from the same side as the detection objective, thereby eliminating limits on lateral dimensions without sacrificing the imaging resolution, depth, and speed. We present a detailed characterization of LSTM, and demonstrate its complementary advantages over LSM for rapid high-resolution quantitative imaging of large intact samples with high uniform quality. The reported LSTM approach is a significant step for the rapid high-resolution quantitative mapping of the structure and function of very large biological systems, such as a clarified thick coronal slab of human brain and uniformly expanded tissues, and also for rapid volumetric calcium imaging of highly motile animals, such as Hydra, undergoing non-isomorphic body shape changes.

Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

PubMed

Ma, Qian; Khademhosseinieh, Bahar; Huang, Eric; Qian, Haoliang; Bakowski, Malina A; Troemel, Emily R; Liu, Zhaowei

2016-08-16

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume.

STED super-resolution microscopy of clinical paraffin-embedded human rectal cancer tissue.

PubMed

Ilgen, Peter; Stoldt, Stefan; Conradi, Lena-Christin; Wurm, Christian Andreas; Rüschoff, Josef; Ghadimi, B Michael; Liersch, Torsten; Jakobs, Stefan

2014-01-01

Formalin fixed and paraffin-embedded human tissue resected during cancer surgery is indispensable for diagnostic and therapeutic purposes and represents a vast and largely unexploited resource for research. Optical microscopy of such specimen is curtailed by the diffraction-limited resolution of conventional optical microscopy. To overcome this limitation, we used STED super-resolution microscopy enabling optical resolution well below the diffraction barrier. We visualized nanoscale protein distributions in sections of well-annotated paraffin-embedded human rectal cancer tissue stored in a clinical repository. Using antisera against several mitochondrial proteins, STED microscopy revealed distinct sub-mitochondrial protein distributions, suggesting a high level of structural preservation. Analysis of human tissues stored for up to 17 years demonstrated that these samples were still amenable for super-resolution microscopy. STED microscopy of sections of HER2 positive rectal adenocarcinoma revealed details in the surface and intracellular HER2 distribution that were blurred in the corresponding conventional images, demonstrating the potential of super-resolution microscopy to explore the thus far largely untapped nanoscale regime in tissues stored in biorepositories.

STED Super-Resolution Microscopy of Clinical Paraffin-Embedded Human Rectal Cancer Tissue

PubMed Central

Wurm, Christian Andreas; Rüschoff, Josef; Ghadimi, B. Michael; Liersch, Torsten; Jakobs, Stefan

2014-01-01

Formalin fixed and paraffin-embedded human tissue resected during cancer surgery is indispensable for diagnostic and therapeutic purposes and represents a vast and largely unexploited resource for research. Optical microscopy of such specimen is curtailed by the diffraction-limited resolution of conventional optical microscopy. To overcome this limitation, we used STED super-resolution microscopy enabling optical resolution well below the diffraction barrier. We visualized nanoscale protein distributions in sections of well-annotated paraffin-embedded human rectal cancer tissue stored in a clinical repository. Using antisera against several mitochondrial proteins, STED microscopy revealed distinct sub-mitochondrial protein distributions, suggesting a high level of structural preservation. Analysis of human tissues stored for up to 17 years demonstrated that these samples were still amenable for super-resolution microscopy. STED microscopy of sections of HER2 positive rectal adenocarcinoma revealed details in the surface and intracellular HER2 distribution that were blurred in the corresponding conventional images, demonstrating the potential of super-resolution microscopy to explore the thus far largely untapped nanoscale regime in tissues stored in biorepositories. PMID:25025184

Compact, light-weight and cost-effective microscope based on lensless incoherent holography for telemedicine applications.

PubMed

Mudanyali, Onur; Tseng, Derek; Oh, Chulwoo; Isikman, Serhan O; Sencan, Ikbal; Bishara, Waheb; Oztoprak, Cetin; Seo, Sungkyu; Khademhosseini, Bahar; Ozcan, Aydogan

2010-06-07

Despite the rapid progress in optical imaging, most of the advanced microscopy modalities still require complex and costly set-ups that unfortunately limit their use beyond well equipped laboratories. In the meantime, microscopy in resource-limited settings has requirements significantly different from those encountered in advanced laboratories, and such imaging devices should be cost-effective, compact, light-weight and appropriately accurate and simple to be usable by minimally trained personnel. Furthermore, these portable microscopes should ideally be digitally integrated as part of a telemedicine network that connects various mobile health-care providers to a central laboratory or hospital. Toward this end, here we demonstrate a lensless on-chip microscope weighing approximately 46 grams with dimensions smaller than 4.2 cm x 4.2 cm x 5.8 cm that achieves sub-cellular resolution over a large field of view of approximately 24 mm(2). This compact and light-weight microscope is based on digital in-line holography and does not need any lenses, bulky optical/mechanical components or coherent sources such as lasers. Instead, it utilizes a simple light-emitting-diode (LED) and a compact opto-electronic sensor-array to record lensless holograms of the objects, which then permits rapid digital reconstruction of regular transmission or differential interference contrast (DIC) images of the objects. Because this lensless incoherent holographic microscope has orders-of-magnitude improved light collection efficiency and is very robust to mechanical misalignments it may offer a cost-effective tool especially for telemedicine applications involving various global health problems in resource limited settings.

Capturing the Surface Texture and Shape of Pollen: A Comparison of Microscopy Techniques

PubMed Central

Sivaguru, Mayandi; Mander, Luke; Fried, Glenn; Punyasena, Surangi W.

2012-01-01

Research on the comparative morphology of pollen grains depends crucially on the application of appropriate microscopy techniques. Information on the performance of microscopy techniques can be used to inform that choice. We compared the ability of several microscopy techniques to provide information on the shape and surface texture of three pollen types with differing morphologies. These techniques are: widefield, apotome, confocal and two-photon microscopy (reflected light techniques), and brightfield and differential interference contrast microscopy (DIC) (transmitted light techniques). We also provide a first view of pollen using super-resolution microscopy. The three pollen types used to contrast the performance of each technique are: Croton hirtus (Euphorbiaceae), Mabea occidentalis (Euphorbiaceae) and Agropyron repens (Poaceae). No single microscopy technique provided an adequate picture of both the shape and surface texture of any of the three pollen types investigated here. The wavelength of incident light, photon-collection ability of the optical technique, signal-to-noise ratio, and the thickness and light absorption characteristics of the exine profoundly affect the recovery of morphological information by a given optical microscopy technique. Reflected light techniques, particularly confocal and two-photon microscopy, best capture pollen shape but provide limited information on very fine surface texture. In contrast, transmitted light techniques, particularly differential interference contrast microscopy, can resolve very fine surface texture but provide limited information on shape. Texture comprising sculptural elements that are spaced near the diffraction limit of light (∼250 nm; NDL) presents an acute challenge to optical microscopy. Super-resolution structured illumination microscopy provides data on the NDL texture of A. repens that is more comparable to textural data from scanning electron microscopy than any other optical microscopy technique investigated here. Maximizing the recovery of morphological information from pollen grains should lead to more robust classifications, and an increase in the taxonomic precision with which ancient vegetation can be reconstructed. PMID:22720050

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

PubMed Central

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

2015-01-01

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

Imaging of single retinal ganglion cell with differential interference contrast microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Oh, Juyeong; Kim, Yu Jeong; Kim, Chul-Ki; Lee, Taik Jin; Seo, Mina; Lee, Seok; Woo, Deok Ha; Jun, Seong Chan; Park, Ki-Ho; Kim, Seok Hwan; Kim, Jae Hun

2017-02-01

Glaucoma is a progressive optic neuropathy, characterized by the selective loss of retinal ganglion cells (RGCs). Therefore, monitoring the change of number or morphology of RGC is essential for the early detection as well as investigation of pathophysiology of glaucoma. Since RGC layer is transparent and hyporeflective, the direct optical visualization of RGCs has not been successful so far. Therefore, glaucoma evaluation mostly depends on indirect diagnostic methods such as the evaluation of optic disc morphology or retinal nerve fiber layer thickness measurement by optical coherence tomography. We have previously demonstrated single photoreceptor cell imaging with differential interference contrast (DIC) microscopy. Herein, we successfully visualized single RGC using DIC microscopy. Since RGC layer is much less reflective than photoreceptor layer, various techniques including the control of light wavelength and bandwidth using a tunable band pass filter were adopted to reduce the chromatic aberration in z-axis for higher and clearer resolution. To verify that the imaged cells were the RGCs, the flat-mounted retina of Sprague-Dawley rat, in which the RGCs were retrogradely labeled with fluorescence, was observed by both fluorescence and DIC microscopies for direct comparison. We have confirmed that the cell images obtained by fluorescence microscopy were perfectly matched with cell images by DIC microscopy. As conclusion, we have visualized single RGC with DIC microscopy, and confirmed with fluorescence microscopy.

Coherent nonlinear optical imaging: beyond fluorescence microscopy.

PubMed

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

2011-01-01

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

Full-field optical coherence microscopy is a novel technique for imaging enteric ganglia in the gastrointestinal tract

PubMed Central

CORON, E.; AUKSORIUS, E.; PIERETTI, A.; MAHÉ, M. M.; LIU, L.; STEIGER, C.; BROMBERG, Y.; BOUMA, B.; TEARNEY, G.; NEUNLIST, M.; GOLDSTEIN, A. M.

2013-01-01

Background Noninvasive methods are needed to improve the diagnosis of enteric neuropathies. Full-field optical coherence microscopy (FFOCM) is a novel optical microscopy modality that can acquire 1 μm resolution images of tissue. The objective of this research was to demonstrate FFOCM imaging for the characterization of the enteric nervous system (ENS). Methods Normal mice and EdnrB−/− mice, a model of Hirschsprung’s disease (HD), were imaged in three-dimensions ex vivo using FFOCM through the entire thickness and length of the gut. Quantitative analysis of myenteric ganglia was performed on FFOCM images obtained from whole-mount tissues and compared with immunohistochemistry imaged by confocal microscopy. Key Results Full-field optical coherence microscopy enabled visualization of the full thickness gut wall from serosa to mucosa. Images of the myenteric plexus were successfully acquired from the stomach, duodenum, colon, and rectum. Quantification of ganglionic neuronal counts on FFOCM images revealed strong interobserver agreement and identical values to those obtained by immunofluorescence microscopy. In EdnrB−/− mice, FFOCM analysis revealed a significant decrease in ganglia density along the colorectum and a significantly lower density of ganglia in all colorectal segments compared with normal mice. Conclusions & Inferences Full-field optical coherence microscopy enables optical microscopic imaging of the ENS within the bowel wall along the entire intestine. FFOCM is able to differentiate ganglionic from aganglionic colon in a mouse model of HD, and can provide quantitative assessment of ganglionic density. With further refinements that enable bowel wall imaging in vivo, this technology has the potential to revolutionize the characterization of the ENS and the diagnosis of enteric neuropathies. PMID:23106847

Pure optical photoacoustic microscopy

PubMed Central

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

2011-01-01

The concept of pure optical photoacoustic microscopy(POPAM) was proposed based on optical rastering of a focused excitation beam and optically sensing the photoacoustic signal using a microring resonator fabricated by a nanoimprinting technique. After the refinements of the microring’s working wavelength and in the resonator structure and mold fabrication, an ultrahigh Q factor of 3.0×105 was achieved which provided high sensitivity with a noise equivalent detectable pressure(NEDP) value of 29Pa. This NEDP is much lower than the hundreds of Pascals achieved with existing optical resonant structures such as etalons, fiber gratings and dielectric multilayer interference filters available for acoustic measurement. The featured high sensitivity allowed the microring resonator to detect the weak photoacoustic signals from micro- or submicroscale objects. The inherent superbroad bandwidth of the optical microring resonator combined with an optically focused scanning beam provided POPAM with high resolution in the axial as well as both lateral directions while the axial resolution of conventional photoacoustic microscopy (PAM) suffers from the limited bandwidth of PZT detectors. Furthermore, the broadband microring resonator showed similar sensitivity to that of our most sensitive PZT detector. The current POPAM system provides a lateral resolution of 5 μm and an axial resolution of 8 μm, comparable to that achieved by optical microscopy while presenting the unique contrast of optical absorption and functional information complementing other optical modalities. The 3D structure of microvasculature, including capillary networks, and even individual red blood cells have been discerned successfully in the proof-of-concept experiments on mouse bladders ex vivo and mouse ears in vivo. The potential of approximately GHz bandwidth of the microring resonator also might allow much higher resolution than shown here in microscopy of optical absorption and acoustic propagation properties at depths in unfrozen tissue specimens or thicker tissue sections, which is not now imageable with current optical or acoustic microscopes of comparable resolution. PMID:21643156

Optical studies of quantum confined nanostructures

NASA Astrophysics Data System (ADS)

Vamivakas, Anthony Nickolas

Recent advances in material growth techniques have led to the laboratory realization of quantum confined nanostructures. By engineering the geometry of these systems it is possible to tailor their optical, electrical and vibrational properties. We now envision integrated electronic and optical devices potentially harnessing quantum mechanical properties of photons, electrons or even phonons. The realization of these next generation devices requires parallel advances in both electrical and optical characterization techniques. In this dissertation we study the optical properties of both zero-dimensional (0D) InAs/GaAs semiconductor quantum dots (QDs) and one-dimensional (1D) single wall carbon nanotubes (SWNTs). We utilize high resolution optical microscopy and spectroscopy techniques to experimentally study both individual QDs and SWNTs. The effect of quantum confinement on light-matter interaction in SWNTs is theoretically investigated. InAs QDs grown by Stranski-Krastanow self-assembly are buried in a GaAs matrix. The planar barriers presented by the dielectric boundary between the GaAs and the host medium limits the optical access to the InAs QDs. Incorporating a numerical aperture increasing microlens (NAIL) into a fiber-based confocal microscope we demonstrate improved ability to couple photons to and from a single InAs QD. With such immersion lens techniques we measure a record 12% extinction of a far-field laser by a single InAs QD. Even typical QD extinction of 6% is visible using a dc power-meter without the need for phase sensitive lock-in detection. This experimental advance will make possible the study of single QDs interacting with engineered vector laser beams. In the optical characterization of SWNTs, one-phonon resonant Raman scattering is employed to measure a tube's electronic resonances and determine the physical diameter and chirality of the tube under study. Recent work has determined excitons dominate the optical response of semiconducting SWNTs. We develop a theory to model the exciton mediated resonant Raman scattering cross-section from a 1D system looking for excitonic signatures in the scattering line shape. Additionally, we theoretically study phonon confinement to a 1D SWNT and use these results to extract the electron-phonon coupling in SWNTs from our Raman measurements. Knowledge of the electron-phonon coupling is a crucial piece of information to characterize a SWNTs electrical transport properties.

Optical sectioning microscopes with no moving parts using a micro-stripe array light emitting diode.

PubMed

Poher, V; Zhang, H X; Kennedy, G T; Griffin, C; Oddos, S; Gu, E; Elson, D S; Girkin, M; French, P M W; Dawson, M D; Neil, M A

2007-09-03

We describe an optical sectioning microscopy system with no moving parts based on a micro-structured stripe-array light emitting diode (LED). By projecting arbitrary line or grid patterns onto the object, we are able to implement a variety of optical sectioning microscopy techniques such as grid-projection structured illumination and line scanning confocal microscopy, switching from one imaging technique to another without modifying the microscope setup. The micro-structured LED and driver are detailed and depth discrimination capabilities are measured and calculated.

Single-Shot Optical Sectioning Using Two-Color Probes in HiLo Fluorescence Microscopy

PubMed Central

Muro, Eleonora; Vermeulen, Pierre; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit; Fragola, Alexandra; Loriette, Vincent

2011-01-01

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate. PMID:21641327

4Pi Microscopy.

PubMed

Schmidt, Roman; Engelhardt, Johann; Lang, Marion

2013-01-01

Optical microscopy has become a key technology in the life sciences today. Its noninvasive nature provides access to the interior of intact and even living cells, where specific molecules can be precisely localized by fluorescent tagging. However, the attainable 3D resolution of an optical microscope has long been hampered by a comparatively poor resolution along the optic axis. By coherent focusing through two objective lenses, 4Pi microscopy improves the axial resolution by three- to fivefold. This primer is intended as a starting point for the design and operation of a 4Pi microscope of type A.

Soft x-ray microscopy and extreme ultraviolet lithography: Imaging in the 20-50 nm regime (abstract) (invited)

NASA Astrophysics Data System (ADS)

Attwood, David

2002-03-01

Advances in short wavelength optics, covering the range from 1 to 14 nm, are providing new results and new opportunities. Zone plate lenses [E. Anderson et al., J. Vac. Sci. Techno. B 18, 2970 (2000)] for soft x-ray microscopy [G. Denbeaux, Rev. Sci. Instrum. (these proceedings); W. Chao, Proc. SPIE 4146, 171 (2000)] are now made to high accuracy with outer zone widths of 25 nm, and demonstrated resolution of 23 nm with proper illumination and stability. These permit important advances in the study of protein specific transport and structure in the life sciences [C. Larabell (private communication); W. Meyer-Ilse et al., J. Microsc. 201, 395 (2001)] and the study of magnetic materials [P. Fischer et al., J. Synchrotron. Radiat. 8, 325 (2001)] with elemental sensitivity at the resolution of individual domains. Major corporations (members of the EUV Limited Liability Company are Intel, Motorola, AMD, Micron, Infineon, and IBM) are now preparing the path for the fabrication of future computer chips, in the years 2007 and beyond, using multilayer coated reflective optics, which achieve reflectivities of 70% in the 11-14 nm region [T. Barbee et al., Appl. Opt. 24, 883 (1985); C. Montcalm et al., Proc. SPIE 3676, 710 (1999)]. These coated optics are to be incorporated in extreme ultraviolet (EUV) print cameras, known as "steppers." Electronic patterns with features in the range of 50-70 nm have been printed. The first alpha tool stepper recently demonstrated all critical technologies [D. Tichenor et al., Proc. SPIE 4343, 19 (2001)] needed for EUV lithography. Preproduction beta tools are targeted for delivery by leading suppliers [ASML, the Netherlands, at the SPIE Microlithography Conference, Santa Clara, CA, March 2001] in 2004, with high volume production tools available in late 2006 for manufacturing in 2007. New results in these two areas will be discussed in the context of the synergy of science and technology.

New frontier in hypericin-mediated diagnosis of cancer with current optical technologies.

PubMed

Olivo, Malini; Fu, Chit Yaw; Raghavan, Vijaya; Lau, Weber Kam On

2012-02-01

Photosensitizers (PSs) have shown great potentials as molecular contrast agents in photodynamic diagnosis (PDD) of cancer. While the diagnostic values of PSs have been proven previously, little efforts have been put into developing optical imaging and diagnostic algorithms. In this article, we review the recent development of optical probes that have been used in conjunction with a potent PS, hypericin (HY). Various fluorescence techniques such as laser confocal microscopy, fluorescence urine cytology, endoscopy and endomicroscopy are covered. We will also discuss about image processing and classification approaches employed for accurate PDD. We anticipate that continual efforts in these developments could lead to an objective PDD and complete surgical clearance of tumors. Recent advancements in nanotechnology have also opened new horizons for PSs. The use of biocompatible gold nanoparticles as carrier for enhanced targeted delivery of HY has been attained. In addition, plasmonic properties of nanoparticles were harnessed to induce localized hyperthermia and to manage the release of PS molecules, enabling a better therapeutic outcome of a combined photodynamic and photothermal therapy. Finally, we discuss how nanoparticles can be used as contrast agents for other optical techniques such as optical coherence tomography and surface-enhanced Raman scattering imaging.

Quantitative X-ray Differential Interference Contrast Microscopy

NASA Astrophysics Data System (ADS)

Nakamura, Takashi

Full-field soft x-ray microscopes are widely used in many fields of sciences. Advances in nanofabrication technology enabled short wavelength focusing elements with significantly improved spatial resolution. In the soft x-ray spectral region, samples as small as 12 nm can be resolved using micro zone-plates as the objective lens. In addition to conventional x-ray microscopy in which x-ray absorption difference provides the image contrast, phase contrast mechanisms such as differential phase contrast (DIC) and Zernike phase contrast have also been demonstrated These phase contrast imaging mechanisms are especially attractive at the x-ray wavelengths where phase contrast of most materials is typically 10 times stronger than the absorption contrast. With recent progresses in plasma-based x- ray sources and increasing accessibility to synchrotron user facilities, x-ray microscopes are quickly becoming standard measurement equipment in the laboratory. To further the usefulness of x-ray DIC microscopy this thesis explicitly addresses three known issues with this imaging modality by introducing new techniques and devices First, as opposed to its visible-light counterpart, no quantitative phase imaging technique exists for x-ray DIC microscopy. To address this issue, two nanoscale x-ray quantitative phase imaging techniques, using exclusive OR (XOR) patterns and zone-plate doublets, respectively, are proposed. Unlike existing x-ray quantitative phase imaging techniques such as Talbot interferometry and ptychography, no dedicated experimental setups or stringent illumination coherence are needed for quantitative phase retrieval. Second, to the best of our knowledge, no quantitative performance characterization of DIC microscopy exists to date. Therefore the imaging system's response to sample's spatial frequency is not known In order to gain in-depth understanding of this imaging modality, performance of x-ray DIC microscopy is quantified using modulation transfer function. A new illumination apparatus required for the transfer function analysis under partially coherent illumination is also proposed. Such a characterization is essential for a proper selection of DIC optics for various transparent samples under study. Finally, optical elements used for x-ray DIC microscopy are highly absorptive and high brilliance x-ray sources such as synchrotrons are generally needed for image contrast. To extend the use of x-ray DIC microscopy to a wider variety of applications, a high efficiency large numerical aperture optical element consisting of high reflective Bragg reflectors is proposed. Using Bragg reflectors, which have 70% ˜99% reflectivity at extreme ultraviolet and soft x-rays for all angles of glancing incidence, the first order focusing efficiency is expected to increase by ˜ 8 times compared to that of a typical Fresnel zone-plate. This thesis contributes to current nanoscale x-ray phase contrast imaging research and provides new insights for biological, material, and magnetic sciences

Measuring Roughnesses Of Optical Surfaces

NASA Technical Reports Server (NTRS)

Coulter, Daniel R.; Al-Jumaily, Gahnim A.; Raouf, Nasrat A.; Anderson, Mark S.

1994-01-01

Report discusses use of scanning tunneling microscopy and atomic force microscopy to measure roughnesses of optical surfaces. These techniques offer greater spatial resolution than other techniques. Report notes scanning tunneling microscopes and atomic force microscopes resolve down to 1 nm.

Nanodiamond Landmarks for Subcellular Multimodal Optical and Electron Imaging

PubMed Central

Zurbuchen, Mark A.; Lake, Michael P.; Kohan, Sirus A.; Leung, Belinda; Bouchard, Louis-S.

2013-01-01

There is a growing need for biolabels that can be used in both optical and electron microscopies, are non-cytotoxic, and do not photobleach. Such biolabels could enable targeted nanoscale imaging of sub-cellular structures, and help to establish correlations between conjugation-delivered biomolecules and function. Here we demonstrate a sub-cellular multi-modal imaging methodology that enables localization of inert particulate probes, consisting of nanodiamonds having fluorescent nitrogen-vacancy centers. These are functionalized to target specific structures, and are observable by both optical and electron microscopies. Nanodiamonds targeted to the nuclear pore complex are rapidly localized in electron-microscopy diffraction mode to enable “zooming-in” to regions of interest for detailed structural investigations. Optical microscopies reveal nanodiamonds for in-vitro tracking or uptake-confirmation. The approach is general, works down to the single nanodiamond level, and can leverage the unique capabilities of nanodiamonds, such as biocompatibility, sensitive magnetometry, and gene and drug delivery. PMID:24036840

Time-resolved wide-field optically sectioned fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dupuis, Guillaume; Benabdallah, Nadia; Chopinaud, Aurélien; Mayet, Céline; Lévêque-Fort, Sandrine

2013-02-01

We present the implementation of a fast wide-field optical sectioning technique called HiLo microscopy on a fluorescence lifetime imaging microscope. HiLo microscopy is based on the fusion of two images, one with structured illumination and another with uniform illumination. Optically sectioned images are then digitally generated thanks to a fusion algorithm. HiLo images are comparable in quality with confocal images but they can be acquired faster over larger fields of view. We obtain 4D imaging by combining HiLo optical sectioning, time-gated detection, and z-displacement. We characterize the performances of this set-up in terms of 3D spatial resolution and time-resolved capabilities in both fixed- and live-cell imaging modes.

Phase contrast and DIC instrumentation and applications in cell, developmental, and marine biology

NASA Astrophysics Data System (ADS)

Gundlach, Heinz

1994-05-01

Nomarski's differential interference contrast (DIC) microscopy is discussed in comparison to Zernike's phase contrast (PhC) microscopy. The possibilities and limits of both are demonstrated by various applications. The high contrast and the use of the full numerical aperture of the DIC optics makes it possible to obtain a series of 'optical sections' through rather thick living specimens (e.g. head of water flea, salivary gland of Drosophila, Xenopus nucleolus, sea urchen egg, mouse embryo). PhC and DIC optics are today available for high resolution light microscopy until N.A. 1.4 Oil as well as for long working distance (LWD) optics, mainly combined with inverted biological microscopes.

Microstructural features of friction stir welded dissimilar Aluminium alloys AA2219-AA7475

NASA Astrophysics Data System (ADS)

Zaman Khan, Noor; Ubaid, Mohammed; Siddiquee, Arshad Noor; Khan, Zahid A.; Al-Ahmari, Abdulrahman; Chen, Xizhang; Haider Abidi, Mustufa

2018-05-01

High strength, good corrosion resistance, light weight make aluminium alloys a material of choice in many industrial sectors like aerospace, marine etc. Problems associated with welding of these alloys by fusion welding processes restricted their use in various industries. Friction stir welding (FSW), a clean solid-state joining process, easily overcomes various difficulties encountered during conventional fusion welding processes. In the present work, the effect of rotational speed (710 rpm, 900 rpm and 1120 rpm) on micro-hardness distribution and microstructure of FSWed dissimilar aluminium alloy joints were analyzed. Plates of AA7475-T761 and AA2219-O having thickness of 2.5 mm were welded by fixing AA7475 on retreating side (RS) and AA2219 on advancing side (AS). Welded joints were characterized by Vickers micro-hardness testing, scanning electron microscopy (SEM) and optical microscopy (OM). Results revealed that rotational speed significantly affects the micro-hardness due to increase in grain size, coarsening and dissolution of strengthening precipitates and re-precipitation. Higher micro-hardness values were observed in stir zone due to grain refinement and re-precipitation. Minimum micro-hardness value was observed at the TMAZ/HAZ of advancing side due to thermal softening.

Silicon nanowires: where mechanics and optics meet at the nanoscale.

PubMed

Ramos, Daniel; Gil-Santos, Eduardo; Malvar, Oscar; Llorens, Jose M; Pini, Valerio; San Paulo, Alvaro; Calleja, Montserrat; Tamayo, Javier

2013-12-06

Mechanical transducers based on nanowires promise revolutionary advances in biological sensing and force microscopy/spectroscopy. A crucial step is the development of simple and non-invasive techniques able to detect displacements with subpicometer sensitivity per unit bandwidth. Here, we design suspended tapered silicon nanowires supporting a range of optical resonances that confine and efficiently scatter light in the visible range. Then, we develop an optical method for efficiently coupling the evanescent field to the regular interference pattern generated by an incoming laser beam and the reflected beam from the substrate underneath the nanowire. This optomechanical coupling is here applied to measure the displacement of 50 nm wide nanowires with sensitivity on the verge of 1 fm/Hz(1/2) at room temperature with a simple laser interferometry set-up. This method opens the door to the measurement of the Brownian motion of ultrashort nanowires for the detection of single biomolecular recognition events in liquids, and single molecule spectroscopy in vacuum.

In vivo imaging of oral neoplasia using a miniaturized fiber optic confocal reflectance microscope.

PubMed

Maitland, Kristen C; Gillenwater, Ann M; Williams, Michelle D; El-Naggar, Adel K; Descour, Michael R; Richards-Kortum, Rebecca R

2008-11-01

The purpose of this study was to determine whether in vivo images of oral mucosa obtained with a fiber optic confocal reflectance microscope could be used to differentiate normal and neoplastic tissues. We imaged 20 oral sites in eight patients undergoing surgery for squamous cell carcinoma. Normal and abnormal areas within the oral cavity were identified clinically, and real-time videos of each site were obtained in vivo using a fiber optic confocal reflectance microscope. Following imaging, each site was biopsied and submitted for histopathologic examination. We identified distinct features, such as nuclear irregularity and spacing, which can be used to qualitatively differentiate between normal and abnormal tissue. Representative confocal images of normal, pre-neoplastic, and neoplastic oral tissue are presented. Previous work using much larger microscopes has demonstrated the ability of confocal reflectance microscopy to image cellular and tissue architecture in situ. New advances in technology have enabled miniaturization of imaging systems for in vivo use.

Optical sectioning in wide-field microscopy obtained by dynamic structured light illumination and detection based on a smart pixel detector array.

PubMed

Mitić, Jelena; Anhut, Tiemo; Meier, Matthias; Ducros, Mathieu; Serov, Alexander; Lasser, Theo

2003-05-01

Optical sectioning in wide-field microscopy is achieved by illumination of the object with a continuously moving single-spatial-frequency pattern and detecting the image with a smart pixel detector array. This detector performs an on-chip electronic signal processing that extracts the optically sectioned image. The optically sectioned image is directly observed in real time without any additional postprocessing.

Microscopy imaging system and method employing stimulated raman spectroscopy as a contrast mechanism

DOEpatents

Xie, Xiaoliang Sunney [Lexington, MA; Freudiger, Christian [Boston, MA; Min, Wei [Cambridge, MA

2011-09-27

A microscopy imaging system includes a first light source for providing a first train of pulses at a first center optical frequency .omega..sub.1, a second light source for providing a second train of pulses at a second center optical frequency .omega..sub.2, a modulator system, an optical detector, and a processor. The modulator system is for modulating a beam property of the second train of pulses at a modulation frequency f of at least 100 kHz. The optical detector is for detecting an integrated intensity of substantially all optical frequency components of the first train of pulses from the common focal volume by blocking the second train of pulses being modulated. The processor is for detecting, a modulation at the modulation frequency f, of the integrated intensity of the optical frequency components of the first train of pulses to provide a pixel of an image for the microscopy imaging system.

Identification of nodal tissue in the living heart using rapid scanning fiber-optics confocal microscopy and extracellular fluorophores.

PubMed

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

2013-09-01

Risks associated with pediatric reconstructive heart surgery include injury of the sinoatrial node (SAN) and atrioventricular node (AVN), requiring cardiac rhythm management using implantable pacemakers. These injuries are the result of difficulties in identifying nodal tissues intraoperatively. Here we describe an approach based on confocal microscopy and extracellular fluorophores to quantify tissue microstructure and identify nodal tissue. Using conventional 3-dimensional confocal microscopy we investigated the microstructural arrangement of SAN, AVN, and atrial working myocardium (AWM) in fixed rat heart. AWM exhibited a regular striated arrangement of the extracellular space. In contrast, SAN and AVN had an irregular, reticulated arrangement. AWM, SAN, and AVN tissues were beneath a thin surface layer of tissue that did not obstruct confocal microscopic imaging. Subsequently, we imaged tissues in living rat hearts with real-time fiber-optics confocal microscopy. Fiber-optics confocal microscopy images resembled images acquired with conventional confocal microscopy. We investigated spatial regularity of tissue microstructure from Fourier analysis and second-order image moments. Fourier analysis of fiber-optics confocal microscopy images showed that the spatial regularity of AWM was greater than that of nodal tissues (37.5 ± 5.0% versus 24.3 ± 3.9% for SAN and 23.8 ± 3.7% for AVN; P<0.05). Similar differences of spatial regularities were revealed from second-order image moments (50.0 ± 7.3% for AWM versus 29.3 ± 6.7% for SAN and 27.3 ± 5.5% for AVN; P<0.05). The study demonstrates feasibility of identifying nodal tissue in living heart using extracellular fluorophores and fiber-optics confocal microscopy. Application of the approach in pediatric reconstructive heart surgery may reduce risks of injuring nodal tissues.

Development of large field-of-view two photon microscopy for imaging mouse cortex (Conference Presentation)

NASA Astrophysics Data System (ADS)

Bumstead, Jonathan; Côté, Daniel C.; Culver, Joseph P.

2017-02-01

Spontaneous neuronal activity has been measured at cellular resolution in mice, zebrafish, and C. elegans using optical sectioning microscopy techniques, such as light sheet microscopy (LSM) and two photon microscopy (TPM). Recent improvements in these modalities and genetically encoded calcium indicators (GECI's) have enabled whole brain imaging of calcium dynamics in zebrafish and C. elegans. However, these whole brain microscopy studies have not been extended to mice due to the limited field of view (FOV) of TPM and the cumbersome geometry of LSM. Conventional TPM is restricted to diffraction limited imaging over this small FOV (around 500 x 500 microns) due to the use of high magnification objectives (e.g. 1.0 NA; 20X) and the aberrations introduced by relay optics used in scanning the beam across the sample. To overcome these limitations, we have redesigned the entire optical path of the two photon microscope (scanning optics and objective lens) to support a field of view of Ø7 mm with relatively high spatial resolution (<10 microns). Using optical engineering software Zemax, we designed our system with commercially available optics that minimize astigmatism, field curvature, chromatic focal shift, and vignetting. Performance of the system was also tested experimentally with fluorescent beads in agarose, fixed samples, and in vivo structural imaging. Our large-FOV TPM provides a modality capable of studying distributed brain networks in mice at cellular resolution.

Wave front engineering by means of diffractive optical elements for applications in microscopy

NASA Astrophysics Data System (ADS)

Cojoc, Dan; Ferrari, Enrico; Garbin, Valeria; Cabrini, Stefano; Carpentiero, Alessandro; Prasciolu, Mauro; Businaro, Luca; Kaulich, Burchard; Di Fabrizio, Enzo

2006-05-01

We present a unified view regarding the use of diffractive optical elements (DOEs) for microscopy applications a wide range of electromagnetic spectrum. The unified treatment is realized through the design and fabrication of DOE through which wave front beam shaping is obtained. In particular we show applications ranging from micromanipulation using optical tweezers to X-ray differential interference contrast (DIC) microscopy. We report some details on the design and physical implementation of diffractive elements that beside focusing perform also other optical functions: beam splitting, beam intensity and phase redistribution or mode conversion. Laser beam splitting is used for multiple trapping and independent manipulation of spherical micro beads and for direct trapping and manipulation of biological cells with non-spherical shapes. Another application is the Gauss to Laguerre-Gaussian mode conversion, which allows to trap and transfer orbital angular momentum of light to micro particles with high refractive index and to trap and manipulate low index particles. These experiments are performed in an inverted optical microscope coupled with an infrared laser beam and a spatial light modulator for DOEs implementation. High resolution optics, fabricated by means of e-beam lithography, are demonstrated to control the intensity and the phase of the sheared beams in X-ray DIC microscopy. DIC experiments with phase objects reveal a dramatic increase in image contrast compared to bright-field X-ray microscopy.

Recent Advances in Fiber Lasers for Nonlinear Microscopy

PubMed Central

Xu, C.; Wise, F. W.

2013-01-01

Nonlinear microscopy techniques developed over the past two decades have provided dramatic new capabilities for biological imaging. The initial demonstrations of nonlinear microscopies coincided with the development of solid-state femtosecond lasers, which continue to dominate applications of nonlinear microscopy. Fiber lasers offer attractive features for biological and biomedical imaging, and recent advances are leading to high-performance sources with the potential for robust, inexpensive, integrated instruments. This article discusses recent advances, and identifies challenges and opportunities for fiber lasers in nonlinear bioimaging. PMID:24416074

Dynamic plasmonic colour display

NASA Astrophysics Data System (ADS)

Duan, Xiaoyang; Kamin, Simon; Liu, Na

2017-02-01

Plasmonic colour printing based on engineered metasurfaces has revolutionized colour display science due to its unprecedented subwavelength resolution and high-density optical data storage. However, advanced plasmonic displays with novel functionalities including dynamic multicolour printing, animations, and highly secure encryption have remained in their infancy. Here we demonstrate a dynamic plasmonic colour display technique that enables all the aforementioned functionalities using catalytic magnesium metasurfaces. Controlled hydrogenation and dehydrogenation of the constituent magnesium nanoparticles, which serve as dynamic pixels, allow for plasmonic colour printing, tuning, erasing and restoration of colour. Different dynamic pixels feature distinct colour transformation kinetics, enabling plasmonic animations. Through smart material processing, information encoded on selected pixels, which are indiscernible to both optical and scanning electron microscopies, can only be read out using hydrogen as a decoding key, suggesting a new generation of information encryption and anti-counterfeiting applications.

Dynamic plasmonic colour display.

PubMed

Duan, Xiaoyang; Kamin, Simon; Liu, Na

2017-02-24

Plasmonic colour printing based on engineered metasurfaces has revolutionized colour display science due to its unprecedented subwavelength resolution and high-density optical data storage. However, advanced plasmonic displays with novel functionalities including dynamic multicolour printing, animations, and highly secure encryption have remained in their infancy. Here we demonstrate a dynamic plasmonic colour display technique that enables all the aforementioned functionalities using catalytic magnesium metasurfaces. Controlled hydrogenation and dehydrogenation of the constituent magnesium nanoparticles, which serve as dynamic pixels, allow for plasmonic colour printing, tuning, erasing and restoration of colour. Different dynamic pixels feature distinct colour transformation kinetics, enabling plasmonic animations. Through smart material processing, information encoded on selected pixels, which are indiscernible to both optical and scanning electron microscopies, can only be read out using hydrogen as a decoding key, suggesting a new generation of information encryption and anti-counterfeiting applications.

Dynamic plasmonic colour display

PubMed Central

Duan, Xiaoyang; Kamin, Simon; Liu, Na

2017-01-01

Plasmonic colour printing based on engineered metasurfaces has revolutionized colour display science due to its unprecedented subwavelength resolution and high-density optical data storage. However, advanced plasmonic displays with novel functionalities including dynamic multicolour printing, animations, and highly secure encryption have remained in their infancy. Here we demonstrate a dynamic plasmonic colour display technique that enables all the aforementioned functionalities using catalytic magnesium metasurfaces. Controlled hydrogenation and dehydrogenation of the constituent magnesium nanoparticles, which serve as dynamic pixels, allow for plasmonic colour printing, tuning, erasing and restoration of colour. Different dynamic pixels feature distinct colour transformation kinetics, enabling plasmonic animations. Through smart material processing, information encoded on selected pixels, which are indiscernible to both optical and scanning electron microscopies, can only be read out using hydrogen as a decoding key, suggesting a new generation of information encryption and anti-counterfeiting applications. PMID:28232722

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.

Laser-combined scanning tunnelling microscopy for probing ultrafast transient dynamics.

PubMed

Terada, Yasuhiko; Yoshida, Shoji; Takeuchi, Osamu; Shigekawa, Hidemi

2010-07-07

The development of time-resolved scanning tunnelling microscopy (STM), in particular, attempts to combine STM with ultrafast laser technology, is reviewed with emphasis on observed physical quantities and spatiotemporal resolution. Ultrashort optical pulse technology has allowed us to observe transient phenomena in the femtosecond range, which, however, has the drawback of a relatively low spatial resolution due to the electromagnetic wavelength used. In contrast, STM and its related techniques, although the time resolution is limited by the circuit bandwidth (∼100 kHz), enable us to observe structures at the atomic level in real space. Our purpose has been to combine these two techniques to achieve a new technology that satisfies the requirements for exploring the ultrafast transient dynamics of the local quantum functions in organized small structures, which will advance the pursuit of future nanoscale scientific research in terms of the ultimate temporal and spatial resolutions. © 2010 IOP Publishing Ltd

Toward investigating changes in cell mechanoelastic properties in response to nanosecond pulsed electric fields

NASA Astrophysics Data System (ADS)

Coker, Zachary; Troyanova-Wood, Maria; Traverso, Andrew; Meng, Zhaokai; Ballmann, Charles; Petrov, Georgi; Ibey, Bennett L.; Yakovlev, Vladislav

2017-02-01

Nanosecond electric pulses (nsEPs) are known to cause a variety of effects on mammalian cells, ranging from destabilization of cell membranes to changes in cytoskeleton and elastic moduli. Measurement of a cells mechanoelastic properties have previously been limited to only invasive and destructive techniques such as atomic force microscopy or application of optical tweezers. However, due to recent advances, Brillouin spectroscopy has now become viable as a non-contact, non-invasive method for measuring these properties in cells and other materials. Here, we present progress toward applying Brillouin spectroscopy using a unique microscopy system for measuring changes in CHO-K1 cells when exposed to nsEPs of 600ns pulse duration with intensity of 50kV/cm. Successful measurement of mechanoelastic changes in these cells will demonstrate Brillouin spectroscopy as a viable method for measuring changes in elastic properties of other cells and living organisms.

Let's push things forward: disruptive technologies and the mechanics of tissue assembly.

PubMed

Varner, Victor D; Nelson, Celeste M

2013-09-01

Although many of the molecular mechanisms that regulate tissue assembly in the embryo have been delineated, the physical forces that couple these mechanisms to actual changes in tissue form remain unclear. Qualitative studies suggest that mechanical loads play a regulatory role in development, but clear quantitative evidence has been lacking. This is partly owing to the complex nature of these problems - embryonic tissues typically undergo large deformations and exhibit evolving, highly viscoelastic material properties. Still, despite these challenges, new disruptive technologies are enabling study of the mechanics of tissue assembly in unprecedented detail. Here, we present novel experimental techniques that enable the study of each component of these physical problems: kinematics, forces, and constitutive properties. Specifically, we detail advances in light sheet microscopy, optical coherence tomography, traction force microscopy, fluorescence force spectroscopy, microrheology and micropatterning. Taken together, these technologies are helping elucidate a more quantitative understanding of the mechanics of tissue assembly.

Microstructures and Microhardness Properties of CMSX-4® Additively Fabricated Through Scanning Laser Epitaxy (SLE)

NASA Astrophysics Data System (ADS)

Basak, Amrita; Holenarasipura Raghu, Shashank; Das, Suman

2017-12-01

Epitaxial CMSX-4® deposition is achieved on CMSX-4® substrates through the scanning laser epitaxy (SLE) process. A thorough analysis is performed using various advanced material characterization techniques, namely high-resolution optical microscopy, scanning electron microscopy, energy-dispersive x-ray spectroscopy, x-ray diffraction, and Vickers microhardness measurements, to characterize and compare the quality of the SLE-fabricated CMSX-4® deposits to the CMSX-4® substrates. The results show that the CMSX-4® deposits have smaller primary dendritic arm spacing, finer γ/ γ' size, weaker elemental segregation, and higher microhardness compared to the investment cast CMSX-4® substrates. The results presented here demonstrate that CMSX-4® is an attractive material for laser-based AM processing and, therefore, can be used in the fabrication of gas turbine hot-section components through AM processing.

A Simple low-cost device enables four epi-illumination techniques on standard light microscopes.

PubMed

Ishmukhametov, Robert R; Russell, Aidan N; Wheeler, Richard J; Nord, Ashley L; Berry, Richard M

2016-02-08

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.

A Simple low-cost device enables four epi-illumination techniques on standard light microscopes

NASA Astrophysics Data System (ADS)

Ishmukhametov, Robert R.; Russell, Aidan N.; Wheeler, Richard J.; Nord, Ashley L.; Berry, Richard M.

2016-02-01

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.

Noninvasive two-photon fluorescence microscopy imaging of mouse retina and RPE through the pupil of the eye

PubMed Central

Palczewska, Grazyna; Dong, Zhiqian; Golczak, Marcin; Hunter, Jennifer J.; Williams, David R.; Alexander, Nathan S.; Palczewski, Krzysztof

2014-01-01

Two-photon excitation microscopy (TPM) can image retinal molecular processes in vivo. Intrinsically fluorescent retinyl esters in sub-cellular structures called retinosomes are an integral part of the visual chromophore regeneration pathway. Fluorescent condensation products of all–trans–retinal accumulate in the eye with age and are also associated with age-related macular degeneration (AMD). Here we report repetitive, dynamic imaging of these compounds in live mice, through the pupil of the eye. Leveraging advanced adaptive optics we developed a data acquisition algorithm that permitted the identification of retinosomes and condensation products in the retinal pigment epithelium (RPE) by their characteristic localization, spectral properties, and absence in genetically modified or drug-treated mice. This imaging approach has the potential to detect early molecular changes in retinoid metabolism that trigger light and AMD-induced retinal defects and to assess the effectiveness of treatments for these conditions. PMID:24952647

Let's push things forward: disruptive technologies and the mechanics of tissue assembly

PubMed Central

Varner, Victor D.; Nelson, Celeste M.

2013-01-01

Although many of the molecular mechanisms that regulate tissue assembly in the embryo have been delineated, the physical forces that couple these mechanisms to actual changes in tissue form remain unclear. Qualitative studies suggest that mechanical loads play a regulatory role in development, but clear quantitative evidence has been lacking. This is partly owing to the complex nature of these problems – embryonic tissues typically undergo large deformations and exhibit evolving, highly viscoelastic material properties. Still, despite these challenges, new disruptive technologies are enabling study of the mechanics of tissue assembly in unprecedented detail. Here, we present novel experimental techniques that enable the study of each component of these physical problems: kinematics, forces, and constitutive properties. Specifically, we detail advances in light sheet microscopy, optical coherence tomography, traction force microscopy, fluorescence force spectroscopy, microrheology and micropatterning. Taken together, these technologies are helping elucidate a more quantitative understanding of the mechanics of tissue assembly. PMID:23907401

Joining of Silicon Carbide: Diffusion Bond Optimization and Characterization

NASA Technical Reports Server (NTRS)

Halbig, Michael C.; Singh, Mrityunjay

2008-01-01

Joining and integration methods are critically needed as enabling technologies for the full utilization of advanced ceramic components in aerospace and aeronautics applications. One such application is a lean direct injector for a turbine engine to achieve low NOx emissions. In the application, several SiC substrates with different hole patterns to form fuel and combustion air channels are bonded to form the injector. Diffusion bonding is a joining approach that offers uniform bonds with high temperature capability, chemical stability, and high strength. Diffusion bonding was investigated with the aid of titanium foils and coatings as the interlayer between SiC substrates to aid bonding. The influence of such variables as interlayer type, interlayer thickness, substrate finish, and processing time were investigated. Optical microscopy, scanning electron microscopy, and electron microprobe analysis were used to characterize the bonds and to identify the reaction formed phases.

Additive Manufacturing of Nickel-Base Superalloy IN100 Through Scanning Laser Epitaxy

NASA Astrophysics Data System (ADS)

Basak, Amrita; Das, Suman

2018-01-01

Scanning laser epitaxy (SLE) is a laser powder bed fusion (LPBF)-based additive manufacturing process that uses a high-power laser to consolidate metal powders facilitating the fabrication of three-dimensional objects. In the present study, SLE is used to produce samples of IN100, a high-γ' non-weldable nickel-base superalloy on similar chemistry substrates. A thorough analysis is performed using various advanced material characterization techniques such as high-resolution optical microscopy, scanning electron microscopy, energy dispersive x-ray spectroscopy, and Vickers microhardness measurements to characterize and compare the quality of the SLE-fabricated IN100 deposits with the investment cast IN100 substrates. The results show that the IN100 deposits have a finer γ/γ' microstructure, weaker elemental segregation, and higher microhardness compared with the substrate. Through this study, it is demonstrated that the SLE process has tremendous potential in the repair and manufacture of gas turbine hot-section components.

Parametric approaches to micro-scale characterization of tissue volumes in vivo and ex vivo: Imaging microvasculature, attenuation, birefringence, and stiffness (Conference Presentation)

NASA Astrophysics Data System (ADS)

Sampson, David D.; Chin, Lixin; Gong, Peijun; Wijesinghe, Philip; Es'haghian, Shaghayegh; Allen, Wesley M.; Klyen, Blake R.; Kirk, Rodney W.; Kennedy, Brendan F.; McLaughlin, Robert A.

2016-03-01

INVITED TALK Advances in imaging tissue microstructure in living subjects, or in freshly excised tissue with minimum preparation and processing, are important for future diagnosis and surgical guidance in the clinical setting, particularly for application to cancer. Whilst microscopy methods continue to advance on the cellular scale and medical imaging is well established on the scale of the whole tumor or organ, it is attractive to consider imaging the tumor environment on the micro-scale, between that of cells and whole tissues. Such a scenario is ideally suited to optical coherence tomography (OCT), with the twin attractions of requiring little or no tissue preparation, and in vivo capability. OCT's intrinsic scattering contrast reveals many morphological features of tumors, but is frequently ineffective in revealing other important aspects, such as microvasculature, or in reliably distinguishing tumor from uninvolved stroma. To address these shortcomings, we are developing several advances on the basic OCT approach. We are exploring speckle fluctuations to image tissue microvasculature and we have been developing several parametric approaches to tissue micro-scale characterization. Our approaches extract, from a three-dimensional OCT data set, a two-dimensional image of an optical parameter, such as attenuation or birefringence, or a mechanical parameter, such as stiffness, that aids in characterizing the tissue. This latter method, termed optical coherence elastography, parallels developments in ultrasound and magnetic resonance imaging. Parametric imaging of birefringence and of stiffness both show promise in addressing the important issue of differentiating cancer from uninvolved stroma in breast tissue.

Recent progress in tissue optical clearing for spectroscopic application

NASA Astrophysics Data System (ADS)

Sdobnov, A. Yu.; Darvin, M. E.; Genina, E. A.; Bashkatov, A. N.; Lademann, J.; Tuchin, V. V.

2018-05-01

This paper aims to review recent progress in optical clearing of the skin and over naturally turbid biological tissues and blood using this technique in vivo and in vitro with multiphoton microscopy, confocal Raman microscopy, confocal microscopy, NIR spectroscopy, optical coherence tomography, and laser speckle contrast imaging. Basic principles of the technique, its safety, advantages and limitations are discussed. The application of optical clearing agent on a tissue allows for controlling the optical properties of tissue. Optical clearing-induced reduction of tissue scattering significantly facilitates the observation of deep-located tissue regions, at the same time improving the resolution and image contrast for a variety of optical imaging methods suitable for clinical applications, such as diagnostics and laser treatment of skin diseases, mucosal tumor imaging, laser disruption of pathological abnormalities, etc. Structural images of different skin layers obtained ex vivo for porcine ear skin samples at application of Omnipaque™ and glycerol solutions during 60 min. Red color corresponds to TPEAF signal channel. Green color corresponds to SHG signal channel.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miranda, Adelaide; De Beule, Pieter A. A., E-mail: pieter.de-beule@inl.int; Martins, Marco

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 discussmore » 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.« less

A correlative optical microscopy and scanning electron microscopy approach to locating nanoparticles in brain tumors.

PubMed

Kempen, Paul J; Kircher, Moritz F; de la Zerda, Adam; Zavaleta, Cristina L; Jokerst, Jesse V; Mellinghoff, Ingo K; Gambhir, Sanjiv S; Sinclair, Robert

2015-01-01

The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Adherens Junctions Revisualized: Organizing Cadherins as Nanoassemblies.

PubMed

Yap, Alpha S; Gomez, Guillermo A; Parton, Robert G

2015-10-12

This Perspective considers how classical cadherin cell-cell adhesion receptors are organized at the nanoscale to generate lateral clusters. Recent advances in optical microscopy reveal that clustering constitutes a general feature of cadherin organization, but one that takes diverse forms. Here we consider the molecular mechanisms responsible for cadherin clustering and their functional implications. We frame our discussion in light of what is known about how nanoscale organization is conferred upon the plasma membrane, through protein-protein interactions, regulation of the cortical actin cytoskeleton, and the lipid environment of the membrane. Copyright © 2015 Elsevier Inc. All rights reserved.

Recent advances in laser in situ keratomileusis-associated dry eye.

PubMed

Xie, Wenjia

2016-03-01

Dry eye is the most common complication after laser in situ keratomileusis (LASIK). The major cause of LASIK-associated dry eye is corneal nerve damage. Early identification and treatment of post-operative dry eye are essential to prevent further ocular surface damage. This article reviews the recent studies of LASIK-associated dry eye, including clinical features, aetiology, risk factors, evaluations and treatment. The applications of novel technologies in LASIK-associated dry eye evaluation like anterior segment spectral-domain optical coherence tomography (SD-OCT) and corneal confocal microscopy are also introduced in this review. © 2016 Optometry Australia.

U-10Mo Sample Preparation and Examination using Optical and Scanning Electron Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prabhakaran, Ramprashad; Joshi, Vineet V.; Rhodes, Mark A.

2016-10-01

The purpose of this document is to provide guidelines to prepare specimens of uranium alloyed with 10 weight percent molybdenum (U-10Mo) for optical metallography and scanning electron microscopy. This document also provides instructions to set up an optical microscope and a scanning electron microscope to analyze U-10Mo specimens and to obtain the required information.

U-10Mo Sample Preparation and Examination using Optical and Scanning Electron Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prabhakaran, Ramprashad; Joshi, Vineet V.; Rhodes, Mark A.

2016-03-30

The purpose of this document is to provide guidelines to prepare specimens of uranium alloyed with 10 weight percent molybdenum (U-10Mo) for optical metallography and scanning electron microscopy. This document also provides instructions to set up an optical microscope and a scanning electron microscope to analyze U-10Mo specimens and to obtain the required information.

Aspects of ultra-high-precision diamond machining of RSA 443 optical aluminium

NASA Astrophysics Data System (ADS)

Mkoko, Z.; Abou-El-Hossein, K.

2015-08-01

Optical aluminium alloys such as 6061-T6 are traditionally used in ultra-high precision manufacturing for making optical mirrors for aerospace and other applications. However, the optics industry has recently witnessed the development of more advanced optical aluminium grades that are capable of addressing some of the issues encountered when turning with single-point natural monocrystalline diamond cutters. The advent of rapidly solidified aluminium (RSA) grades has generally opened up new possibilities for ultra-high precision manufacturing of optical components. In this study, experiments were conducted with single-point diamond cutters on rapidly solidified aluminium RSA 443 material. The objective of this study is to observe the effects of depth of cut and feed rate at a fixed rotational speed on the tool wear rate and resulting surface roughness of diamond turned specimens. This is done to gain further understanding of the rate of wear on the diamond cutters versus the surface texture generated on the RSA 443 material. The diamond machining experiments yielded machined surfaces which are less reflective but with consistent surface roughness values. Cutting tools were observed for wear through scanning microscopy; relatively low wear pattern was evident on the diamond tool edge. The highest tool wear were obtained at higher depth of cut and increased feed rate.

Large scale superres 3D imaging: light-sheet single-molecule localization microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lu, Chieh Han; Chen, Peilin; Chen, Bi-Chang

2017-02-01

Optical imaging techniques provide much important information in understanding life science especially cellular structure and morphology because "seeing is believing". However, the resolution of optical imaging is limited by the diffraction limit, which is discovered by Ernst Abbe, i.e. λ/2(NA) (NA is the numerical aperture of the objective lens). Fluorescence super-resolution microscopic techniques such as Stimulated emission depletion microscopy (STED), Photoactivated localization microscopy (PALM), and Stochastic optical reconstruction microscopy (STORM) are invented to have the capability of seeing biological entities down to molecular level that are smaller than the diffraction limit (around 200-nm in lateral resolution). These techniques do not physically violate the Abbe limit of resolution but exploit the photoluminescence properties and labelling specificity of fluorescence molecules to achieve super-resolution imaging. However, these super-resolution techniques limit most of their applications to the 2D imaging of fixed or dead samples due to the high laser power needed or slow speed for the localization process. Extended from 2D imaging, light sheet microscopy has been proven to have a lot of applications on 3D imaging at much better spatiotemporal resolutions due to its intrinsic optical sectioning and high imaging speed. Herein, we combine the advantage of localization microscopy and light-sheet microscopy to have super-resolved cellular imaging in 3D across large field of view. With high-density labeled spontaneous blinking fluorophore and wide-field detection of light-sheet microscopy, these allow us to construct 3D super-resolution multi-cellular imaging at high speed ( minutes) by light-sheet single-molecule localization microscopy.

Intravital hybrid optical-optoacoustic microscopy based on fiber-Bragg interferometry

NASA Astrophysics Data System (ADS)

Shnaiderman, Rami; Wissmeyer, Georg; Seeger, Markus; Estrada, Hector; Ntziachristos, Vasilis

2018-02-01

Optoacoustic microscopy (OAM) has enabled high-resolution, label-free imaging of tissues at depths not achievable with purely optical microscopy. However, widespread implementation of OAM into existing epi-illumination microscopy setups is often constrained by the performance and size of the commonly used piezoelectric ultrasound detectors. In this work, we introduce a novel acoustic detector based on a π-phase-shifted fiber Bragg grating (π-FBG) interferometer embedded inside an ellipsoidal acoustic cavity. The cavity enables seamless integration of epi-illumination OAM into existing microscopy setups by decoupling the acoustic and optical paths between the microscope objective and the sample. The cavity also acts as an acoustic condenser, boosting the sensitivity of the π-FBG and enabling cost effective CW-laser interrogation technique. We characterize the sensor's sensitivity and bandwidth and demonstrate hybrid OAM and second-harmonic imaging of phantoms and mouse tissue in vivo.

High resolution multiple excitation spot optical microscopy

NASA Astrophysics Data System (ADS)

Dilipkumar, Shilpa; Mondal, Partha Pratim

2011-06-01

We propose fundamental improvements in three-dimensional (3D) resolution of multiple excitation spot optical microscopy. The excitation point spread function (PSF) is generated by two interfering counter-propagating depth-of-focus beams along the optical axis. Detection PSF is obtained by coherently interfering the emitted fluorescent light (collected by both the objectives) at the detector. System PSF shows upto 14-fold reduction in focal volume as compared to confocal, and almost 2-fold improvement in lateral resolution. Proposed PSF has the ability to simultaneously excite multiple 3D-spots of sub-femtoliter volume. Potential applications are in fluorescence microscopy and nanobioimaging.

Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy.

PubMed

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

2015-01-01

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

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

Quantifying structural alterations in Alzheimer's disease brains using quantitative phase imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lee, Moosung; Lee, Eeksung; Jung, JaeHwang; Yu, Hyeonseung; Kim, Kyoohyun; Yoon, Jonghee; Lee, Shinhwa; Jeong, Yong; Park, YongKeun

2017-02-01

Imaging brain tissues is an essential part of neuroscience because understanding brain structure provides relevant information about brain functions and alterations associated with diseases. Magnetic resonance imaging and positron emission tomography exemplify conventional brain imaging tools, but these techniques suffer from low spatial resolution around 100 μm. As a complementary method, histopathology has been utilized with the development of optical microscopy. The traditional method provides the structural information about biological tissues to cellular scales, but relies on labor-intensive staining procedures. With the advances of illumination sources, label-free imaging techniques based on nonlinear interactions, such as multiphoton excitations and Raman scattering, have been applied to molecule-specific histopathology. Nevertheless, these techniques provide limited qualitative information and require a pulsed laser, which is difficult to use for pathologists with no laser training. Here, we present a label-free optical imaging of mouse brain tissues for addressing structural alteration in Alzheimer's disease. To achieve the mesoscopic, unlabeled tissue images with high contrast and sub-micrometer lateral resolution, we employed holographic microscopy and an automated scanning platform. From the acquired hologram of the brain tissues, we could retrieve scattering coefficients and anisotropies according to the modified scattering-phase theorem. This label-free imaging technique enabled direct access to structural information throughout the tissues with a sub-micrometer lateral resolution and presented a unique means to investigate the structural changes in the optical properties of biological tissues.

Tracking individual membrane proteins and their biochemistry: The power of direct observation.

PubMed

Barden, Adam O; Goler, Adam S; Humphreys, Sara C; Tabatabaei, Samaneh; Lochner, Martin; Ruepp, Marc-David; Jack, Thomas; Simonin, Jonathan; Thompson, Andrew J; Jones, Jeffrey P; Brozik, James A

2015-11-01

The advent of single molecule fluorescence microscopy has allowed experimental molecular biophysics and biochemistry to transcend traditional ensemble measurements, where the behavior of individual proteins could not be precisely sampled. The recent explosion in popularity of new super-resolution and super-localization techniques coupled with technical advances in optical designs and fast highly sensitive cameras with single photon sensitivity and millisecond time resolution have made it possible to track key motions, reactions, and interactions of individual proteins with high temporal resolution and spatial resolution well beyond the diffraction limit. Within the purview of membrane proteins and ligand gated ion channels (LGICs), these outstanding advances in single molecule microscopy allow for the direct observation of discrete biochemical states and their fluctuation dynamics. Such observations are fundamentally important for understanding molecular-level mechanisms governing these systems. Examples reviewed here include the effects of allostery on the stoichiometry of ligand binding in the presence of fluorescent ligands; the observation of subdomain partitioning of membrane proteins due to microenvironment effects; and the use of single particle tracking experiments to elucidate characteristics of membrane protein diffusion and the direct measurement of thermodynamic properties, which govern the free energy landscape of protein dimerization. The review of such characteristic topics represents a snapshot of efforts to push the boundaries of fluorescence microscopy of membrane proteins to the absolute limit. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'. Copyright © 2015 Elsevier Ltd. All rights reserved.

Isometric multimodal photoacoustic microscopy based on optically transparent micro-ring ultrasonic detection.

PubMed

Dong, Biqin; Li, Hao; Zhang, Zhen; Zhang, Kevin; Chen, Siyu; Sun, Cheng; Zhang, Hao F

2015-01-01

Photoacoustic microscopy (PAM) is an attractive imaging tool complementary to established optical microscopic modalities by providing additional molecular specificities through imaging optical absorption contrast. While the development of optical resolution photoacoustic microscopy (ORPAM) offers high lateral resolution, the acoustically-determined axial resolution is limited due to the constraint in ultrasonic detection bandwidth. ORPAM with isometric spatial resolution along both axial and lateral direction is yet to be developed. Although recently developed sophisticated optical illumination and reconstruction methods offer improved axial resolution in ORPAM, the image acquisition procedures are rather complicated, limiting their capabilities for high-speed imaging and being easily integrated with established optical microscopic modalities. Here we report an isometric ORPAM based on an optically transparent micro-ring resonator ultrasonic detector and a commercial inverted microscope platform. Owing to the superior spatial resolution and the ease of integrating our ORPAM with established microscopic modalities, single cell imaging with extrinsic fluorescence staining, intrinsic autofluorescence, and optical absorption can be achieved simultaneously. This technique holds promise to greatly improve the accessibility of PAM to the broader biomedical researchers.

Single shot damage mechanism of Mo/Si multilayer optics under intense pulsed XUV-exposure.

PubMed

Khorsand, A R; Sobierajski, R; Louis, E; Bruijn, S; van Hattum, E D; van de Kruijs, R W E; Jurek, M; Klinger, D; Pelka, J B; Juha, L; Burian, T; Chalupsky, J; Cihelka, J; Hajkova, V; Vysin, L; Jastrow, U; Stojanovic, N; Toleikis, S; Wabnitz, H; Tiedtke, K; Sokolowski-Tinten, K; Shymanovich, U; Krzywinski, J; Hau-Riege, S; London, R; Gleeson, A; Gullikson, E M; Bijkerk, F

2010-01-18

We investigated single shot damage of Mo/Si multilayer coatings exposed to the intense fs XUV radiation at the Free-electron LASer facility in Hamburg - FLASH. The interaction process was studied in situ by XUV reflectometry, time resolved optical microscopy, and "post-mortem" by interference-polarizing optical microscopy (with Nomarski contrast), atomic force microscopy, and scanning transmission electron microcopy. An ultrafast molybdenum silicide formation due to enhanced atomic diffusion in melted silicon has been determined to be the key process in the damage mechanism. The influence of the energy diffusion on the damage process was estimated. The results are of significance for the design of multilayer optics for a new generation of pulsed (from atto- to nanosecond) XUV sources.

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.

3D Image Analysis of Geomaterials using Confocal Microscopy

NASA Astrophysics Data System (ADS)

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

2009-05-01

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

Techniques for super-resolution microscopy using NV-diamond

NASA Astrophysics Data System (ADS)

Trifonov, Alexei; Glenn, David; Bar-Gill, Nir; Le Sage, David; Walsworth, Ronald

2011-05-01

We discuss the development and application of techniques for super-resolution microscopy using NV centers in diamond: stimulated emission depletion (STED), metastable ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM). NV centers do not bleach under optical excitation, are not biotoxic, and have long-lived electronic spin coherence and spin-state-dependent fluorescence. Thus NV-diamond has great potential as a fluorescent biomarker and as a magnetic biosensor.

Value of Reflected Light Microscopy in Teaching.

ERIC Educational Resources Information Center

Pasteris, Jill Dill

1983-01-01

Briefly reviews some optical and other physical properties of minerals that can be determined in reflected/incident light. Topics include optical properties of minerals, reflectance, internal reflections, color, bireflectance and reflection pleochroism, anisotropism, zonation, and reflected light microscopy as a teaching tool in undergraduate…

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.

Three-Dimensional Unstained Live-Cell Imaging Using Stimulated Parametric Emission Microscopy

NASA Astrophysics Data System (ADS)

Dang, Hieu M.; Kawasumi, Takehito; Omura, Gen; Umano, Toshiyuki; Kajiyama, Shin'ichiro; Ozeki, Yasuyuki; Itoh, Kazuyoshi; Fukui, Kiichi

2009-09-01

The ability to perform high-resolution unstained live imaging is very important to in vivo study of cell structures and functions. Stimulated parametric emission (SPE) microscopy is a nonlinear-optical microscopy based on ultra-fast electronic nonlinear-optical responses. For the first time, we have successfully applied this technique to archive three-dimensional (3D) images of unstained sub-cellular structures, such as, microtubules, nuclei, nucleoli, etc. in live cells. Observation of a complete cell division confirms the ability of SPE microscopy for long time-scale imaging.

Optically Sectioned Imaging of Microvasculature of In-Vivo and Ex-Vivo Thick Tissue Models with Speckle-illumination HiLo Microscopy and HiLo Image Processing Implementation in MATLAB Architecture

NASA Astrophysics Data System (ADS)

Suen, Ricky Wai

The work described in this thesis covers the conversion of HiLo image processing into MATLAB architecture and the use of speckle-illumination HiLo microscopy for use of ex-vivo and in-vivo imaging of thick tissue models. HiLo microscopy is a wide-field fluorescence imaging technique and has been demonstrated to produce optically sectioned images comparable to confocal in thin samples. The imaging technique was developed by Jerome Mertz and the Boston University Biomicroscopy Lab and has been implemented in our lab as a stand-alone optical setup and a modification to a conventional fluorescence microscope. Speckle-illumination HiLo microscopy combines two images taken under speckle-illumination and standard uniform-illumination to generate an optically sectioned image that reject out-of-focus fluorescence. The evaluated speckle contrast in the images is used as a weighting function where elements that move out-of-focus have a speckle contrast that decays to zero. The experiments shown here demonstrate the capability of our HiLo microscopes to produce optically-sectioned images of the microvasculature of ex-vivo and in-vivo thick tissue models. The HiLo microscope were used to image the microvasculature of ex-vivo mouse heart sections prepared for optical histology and the microvasculature of in-vivo rodent dorsal window chamber models. Studies in label-free surface profiling with HiLo microscopy is also presented.

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. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Adaptive optics stochastic optical reconstruction microscopy (AO-STORM) by particle swarm optimization

PubMed Central

Tehrani, Kayvan F.; Zhang, Yiwen; Shen, Ping; Kner, Peter

2017-01-01

Stochastic optical reconstruction microscopy (STORM) can achieve resolutions of better than 20nm imaging single fluorescently labeled cells. However, when optical aberrations induced by larger biological samples degrade the point spread function (PSF), the localization accuracy and number of localizations are both reduced, destroying the resolution of STORM. Adaptive optics (AO) can be used to correct the wavefront, restoring the high resolution of STORM. A challenge for AO-STORM microscopy is the development of robust optimization algorithms which can efficiently correct the wavefront from stochastic raw STORM images. Here we present the implementation of a particle swarm optimization (PSO) approach with a Fourier metric for real-time correction of wavefront aberrations during STORM acquisition. We apply our approach to imaging boutons 100 μm deep inside the central nervous system (CNS) of Drosophila melanogaster larvae achieving a resolution of 146 nm. PMID:29188105

Adaptive optics stochastic optical reconstruction microscopy (AO-STORM) by particle swarm optimization.

PubMed

Tehrani, Kayvan F; Zhang, Yiwen; Shen, Ping; Kner, Peter

2017-11-01

Stochastic optical reconstruction microscopy (STORM) can achieve resolutions of better than 20nm imaging single fluorescently labeled cells. However, when optical aberrations induced by larger biological samples degrade the point spread function (PSF), the localization accuracy and number of localizations are both reduced, destroying the resolution of STORM. Adaptive optics (AO) can be used to correct the wavefront, restoring the high resolution of STORM. A challenge for AO-STORM microscopy is the development of robust optimization algorithms which can efficiently correct the wavefront from stochastic raw STORM images. Here we present the implementation of a particle swarm optimization (PSO) approach with a Fourier metric for real-time correction of wavefront aberrations during STORM acquisition. We apply our approach to imaging boutons 100 μm deep inside the central nervous system (CNS) of Drosophila melanogaster larvae achieving a resolution of 146 nm.

New approaches in renal microscopy: volumetric imaging and superresolution microscopy.

PubMed

Kim, Alfred H J; Suleiman, Hani; Shaw, Andrey S

2016-05-01

Histologic and electron microscopic analysis of the kidney has provided tremendous insight into structures such as the glomerulus and nephron. Recent advances in imaging, such as deep volumetric approaches and superresolution microscopy, have the capacity to dramatically enhance our current understanding of the structure and function of the kidney. Volumetric imaging can generate images millimeters below the surface of the intact kidney. Superresolution microscopy breaks the diffraction barrier inherent in traditional light microscopy, enabling the visualization of fine structures. Here, we describe new approaches to deep volumetric and superresolution microscopy of the kidney. Rapid advances in lasers, microscopic objectives, and tissue preparation have transformed our ability to deep volumetric image the kidney. Innovations in sample preparation have allowed for superresolution imaging with electron microscopy correlation, providing unprecedented insight into the structures within the glomerulus. Technological advances in imaging have revolutionized our capacity to image both large volumes of tissue and the finest structural details of a cell. These new advances have the potential to provide additional profound observations into the normal and pathologic functions of the kidney.

(Gene sequencing by scanning molecular exciton microscopy)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1991-01-01

This report details progress made in setting up a laboratory for optical microscopy of genes. The apparatus including a fluorescence microscope, a scanning optical microscope, various spectrometers, and supporting computers is described. Results in developing photon and exciton tips, and in preparing samples are presented. (GHH)

Temporal overlap estimation based on interference spectrum in CARS microscopy

NASA Astrophysics Data System (ADS)

Zhang, Yongning; Jiang, Junfeng; Liu, Kun; Huang, Can; Wang, Shuang; Zhang, Xuezhi; Liu, Tiegen

2018-01-01

Coherent Anti-Stokes Raman Scattering (CARS) microscopy has attracted lots of attention because of the advantages, such as noninvasive, label-free, chemical specificity, intrinsic three-dimension spatial resolution and so on. However, the temporal overlap of pump and Stokes has not been solved owing to the ultrafast optical pulse used in CARS microscopy. We combine interference spectrum of residual pump in Stokes path and nonlinear Schrodinger equation (NLSE) to realize the temporal overlap of pump pulse and Stokes pulse. At first, based on the interference spectrum of pump pulse and residual pump in Stokes path, the optical delay is defined when optical path difference between pump path and Stokes path is zero. Then the relative optical delay between Stokes pulse and residual pump in PCF can be calculated by NLSE. According to the spectrum interference and NLSE, temporal overlap of pump pulse and Stokes pulse will be realized easily and the imaging speed will be improved in CARS microscopy.

High speed wavefront sensorless aberration correction in digital micromirror based confocal microscopy.

PubMed

Pozzi, P; Wilding, D; Soloviev, O; Verstraete, H; Bliek, L; Vdovin, G; Verhaegen, M

2017-01-23

The quality of fluorescence microscopy images is often impaired by the presence of sample induced optical aberrations. Adaptive optical elements such as deformable mirrors or spatial light modulators can be used to correct aberrations. However, previously reported techniques either require special sample preparation, or time consuming optimization procedures for the correction of static aberrations. This paper reports a technique for optical sectioning fluorescence microscopy capable of correcting dynamic aberrations in any fluorescent sample during the acquisition. This is achieved by implementing adaptive optics in a non conventional confocal microscopy setup, with multiple programmable confocal apertures, in which out of focus light can be separately detected, and used to optimize the correction performance with a sampling frequency an order of magnitude faster than the imaging rate of the system. The paper reports results comparing the correction performances to traditional image optimization algorithms, and demonstrates how the system can compensate for dynamic changes in the aberrations, such as those introduced during a focal stack acquisition though a thick sample.

Web-based virtual microscopy at the RWTH Aachen University: didactic concept, methods and analysis of acceptance by the students.

PubMed

Merk, Magdalene; Knuechel, Ruth; Perez-Bouza, Alberto

2010-12-20

Fundamental knowledge of microscopic anatomy and pathology has always been an essential part in medical education. The traditional didactic concept comprises theoretical and practical lessons using a light microscope and glass slides. High-speed Internet connections and technical improvement in whole-slide digital microscopy (commonly termed "virtual microscopy") provide a new and attractive approach for both teachers and students. High picture quality and unlimited temporal and spatial availability of histology samples from different fields are key advantages of web-based digital microscopy. In this report we discuss the technical requirements, system efficiency, optical resolution and didactic concept. Furthermore, we present a review of the experience gained in the course of one year based on an analysis of student acceptance. Three groups with a total of 192 students between the 3rd and 5th year of medical studies attending the practical courses of general and advanced histopathology had access to both glass-mounted and digitalized slides. Prior to exams, students were asked to answer an anonymous questionnaire. The results of the study reflect the high acceptance and intensive use of the web-based digital histology by students, thus encouraging the development of further Web-based learning strategies for the teaching of histology and pathology. 2010 Elsevier GmbH. All rights reserved.

Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations

PubMed Central

Szczurek, Aleksander; Klewes, Ludger; Xing, Jun; Gourram, Amine; Birk, Udo; Knecht, Hans; Dobrucki, Jurek W.; Mai, Sabine

2017-01-01

Abstract Advanced light microscopy is an important tool for nanostructure analysis of chromatin. In this report we present a general concept for Single Molecule localization Microscopy (SMLM) super-resolved imaging of DNA-binding dyes based on modifying the properties of DNA and the dye. By careful adjustment of the chemical environment leading to local, reversible DNA melting and hybridization control over the fluorescence signal of the DNA-binding dye molecules can be introduced. We postulate a transient binding as the basis for our variation of binding-activated localization microscopy (BALM). We demonstrate that several intercalating and minor-groove binding DNA dyes can be used to register (optically isolate) only a few DNA-binding dye signals at a time. To highlight this DNA structure fluctuation-assisted BALM (fBALM), we applied it to measure, for the first time, nanoscale differences in nuclear architecture in model ischemia with an anticipated structural resolution of approximately 50 nm. Our data suggest that this approach may open an avenue for the enhanced microscopic analysis of chromatin nano-architecture and hence the microscopic analysis of nuclear structure aberrations occurring in various pathological conditions. It may also become possible to analyse nuclear nanostructure differences in different cell types, stages of development or environmental stress conditions. PMID:28082388

Practical guidelines for implementing adaptive optics in fluorescence microscopy

NASA Astrophysics Data System (ADS)

Wilding, Dean; Pozzi, Paolo; Soloviev, Oleg; Vdovin, Gleb; Verhaegen, Michel

2018-02-01

In life sciences, interest in the microscopic imaging of increasingly complex three dimensional samples, such as cell spheroids, zebrafish embryos, and in vivo applications in small animals, is growing quickly. Due to the increasing complexity of samples, more and more life scientists are considering the implementation of adaptive optics in their experimental setups. While several approaches to adaptive optics in microscopy have been reported, it is often difficult and confusing for the microscopist to choose from the array of techniques and equipment. In this poster presentation we offer a small guide to adaptive optics providing general guidelines for successful adaptive optics implementation.

Through a Window, Brightly: A Review of Selected Nanofabricated Thin-Film Platforms for Spectroscopy, Imaging, and Detection.

PubMed

Dwyer, Jason R; Harb, Maher

2017-09-01

We present a review of the use of selected nanofabricated thin films to deliver a host of capabilities and insights spanning bioanalytical and biophysical chemistry, materials science, and fundamental molecular-level research. We discuss approaches where thin films have been vital, enabling experimental studies using a variety of optical spectroscopies across the visible and infrared spectral range, electron microscopies, and related techniques such as electron energy loss spectroscopy, X-ray photoelectron spectroscopy, and single molecule sensing. We anchor this broad discussion by highlighting two particularly exciting exemplars: a thin-walled nanofluidic sample cell concept that has advanced the discovery horizons of ultrafast spectroscopy and of electron microscopy investigations of in-liquid samples; and a unique class of thin-film-based nanofluidic devices, designed around a nanopore, with expansive prospects for single molecule sensing. Free-standing, low-stress silicon nitride membranes are a canonical structural element for these applications, and we elucidate the fabrication and resulting features-including mechanical stability, optical properties, X-ray and electron scattering properties, and chemical nature-of this material in this format. We also outline design and performance principles and include a discussion of underlying material preparations and properties suitable for understanding the use of alternative thin-film materials such as graphene.

Active microrheology of entangled biopolymer composites link polymer flexibility and length to molecular force response

NASA Astrophysics Data System (ADS)

Fitzpatrick, Robert; Hauer, Cole; Kyrillos, Carl; McGorty, Ryan; Robertson-Anderson, Rae

Entangled polymers have complex viscoelastic properties that are tuned by polymer lengths and flexibilities. Entangled composites of distinct polymers offer added versatility and display nonlinear mechanics, serving as a platform for multifunctional materials. To determine the role of flexibility and length in polymer composites we use optical tweezers and confocal microscopy to measure mechanical and structural properties of co-entangled actin and DNA. Actin filaments have lengths of 5-20 μm, comparable to their persistence length, while DNA of similar lengths have hundreds of persistence lengths per chain. To characterize the nonlinear mechanics of actin-DNA composites, we optically drive a microsphere through the composite and measure the induced force during and following strain. We characterize viscoelasticity and relaxation timescales; and determine the dependence of these quantities on the actin:DNA ratio (0:1-1:0) and DNA length (4-100 μm). We use confocal microscopy to image distinctly labeled co-entangled actin and DNA and characterize network homogeneity and fluctuations. Initial results show actin and DNA are well-integrated and form structurally homogenous networks that exhibit stiffness and relaxation times that increase nonlinearly with increased actin. NSF Career Award (DMR-1254340), AFOSR Young Investigator Program Award (FA95550-12-1-0315), Scialog Collaborative Innovation Award funed by Research Corp. for Scientific Advancement (24192).

Microscale force response and morphology of tunable co-polymerized cytoskeleton networks

NASA Astrophysics Data System (ADS)

Ricketts, Shea; Yadav, Vikrant; Ross, Jennifer L.; Robertson-Anderson, Rae M.

The cytoskeleton is largely comprised of actin and microtubules that entangle and crosslink to form complex networks and structures, giving rise to nonlinear multifunctional mechanics in cells. The relative concentrations of semiflexible actin filaments and rigid microtubules tune cytoskeleton function, allowing cells to move and divide while maintaining rigidity and resilience. To elucidate this complex tunability, we create in vitro composites of co-polymerized actin and microtubules with actin:microtubule molar ratios of 0:1-1:0. We use optical tweezers and confocal microscopy to characterize the nonlinear microscale force response and morphology of the composites. We optically drag a microsphere 30 μm through varying actin-microtubule networks at 10 μm/s and 20 μm/s, and measure the force the networks exerts to resist the strain and the force relaxation following strain. We use dual-color confocal microscopy to image distinctly-labeled filaments in the networks, and characterize the integration of actin and microtubules, network connectivity, and filament rigidity. We find that increasing the fraction of microtubules in networks non-monotonically increases elasticity and stiffness, and hinders force relaxation by suppressing network mobility and fluctuations. NSF CAREER Award (DMR-1255446), Scialog Collaborative Innovation Award funded by Research Corporation for Scientific Advancement (Grant No. 24192).

Developing and Incorporating Instructional Videos and Quizzes as a Blended and Online Learning Component in an Undergraduate Optical Microscopy Curriculum.

NASA Astrophysics Data System (ADS)

Tramontano, S.; Gualda, G. A. R.; Claiborne, L. L.; Brame, C.

2015-12-01

Optical mineralogy is not an easy skill to master as an undergraduate, but it is crucial for understanding what the Earth is made out of. It is a supplementary and specific skillset typically taught in a microscope lab supporting lessons on crystallography, chemistry and mineral analysis in the classroom. Mastering the basic skills is required for advancement in courses that utilize thin sections in teaching igneous, metamorphic, and sedimentary rocks. This project asks: Will exposing undergraduate Earth and environmental studies students to optical microscopy figures in videos prior to lab assist in the acquisition of skills required to describe and distinguish Earth materials? This project is conducted in conjunction with the Blended and Online Learning Design (BOLD) Fellowship offered through the Center for Teaching (CFT) at Vanderbilt University. Eight videos and accompanying pre-lab questions were hosted online weekly in a semester-long, undergraduate Earth materials course. The focus of the design of the videos and supporting questions is specifically on microscopy skills rather than on optics concepts, which is taught post-video. The videos were made available prior to a weekly lab with the intent of familiarizing the student with the types of images and information he/she should obtain with the microscope. Multiple choice, formative-style questions accompany the videos in an online-hosted assignment. These questions are graded on basis of completion and are intended to aid in student metacognition. Subjects include students in the Vanderbilt University Earth Materials course and students from the Hanover College Mineralogy course. The effectiveness of the videos is assessed in two parts: (1) Comparing the homework and lab final grades of the students this year with those of the students last year (2) Analysis of a weekly questionnaire. The answers after each week will be compiled and compared. Collecting data from Vanderbilt University students and Hanover College students diversifies the student population for this study, making it more comprehensive and applicable across different types of institutions. We will use our assessment of the effectiveness of the videos this year to modify and improve the videos and their use in future offerings of the courses.

Full optical model of micro-endoscope with optical coherence microscopy, multiphoton microscopy and visible capabilities

NASA Astrophysics Data System (ADS)

Vega, David; Kiekens, Kelli C.; Syson, Nikolas C.; Romano, Gabriella; Baker, Tressa; Barton, Jennifer K.

2018-02-01

While Optical Coherence Microscopy (OCM), Multiphoton Microscopy (MPM), and narrowband imaging are powerful imaging techniques that can be used to detect cancer, each imaging technique has limitations when used by itself. Combining them into an endoscope to work in synergy can help achieve high sensitivity and specificity for diagnosis at the point of care. Such complex endoscopes have an elevated risk of failure, and performing proper modelling ensures functionality and minimizes risk. We present full 2D and 3D models of a multimodality optical micro-endoscope to provide real-time detection of carcinomas, called a salpingoscope. The models evaluate the endoscope illumination and light collection capabilities of various modalities. The design features two optical paths with different numerical apertures (NA) through a single lens system with a scanning optical fiber. The dual path is achieved using dichroic coatings embedded in a triplet. A high NA optical path is designed to perform OCM and MPM while a low NA optical path is designed for the visible spectrum to navigate the endoscope to areas of interest and narrowband imaging. Different tests such as the reflectance profile of homogeneous epithelial tissue were performed to adjust the models properly. Light collection models for the different modalities were created and tested for efficiency. While it is challenging to evaluate the efficiency of multimodality endoscopes, the models ensure that the system is design for the expected light collection levels to provide detectable signal to work for the intended imaging.

Axial range of conjugate adaptive optics in two-photon microscopy

PubMed Central

Paudel, Hari P.; Taranto, John; Mertz, Jerome; Bifano, Thomas

2015-01-01

We describe an adaptive optics technique for two-photon microscopy in which the deformable mirror used for aberration compensation is positioned in a plane conjugate to the plane of the aberration. We demonstrate in a proof-of-principle experiment that this technique yields a large field of view advantage in comparison to standard pupil-conjugate adaptive optics. Further, we show that the extended field of view in conjugate AO is maintained over a relatively large axial translation of the deformable mirror with respect to the conjugate plane. We conclude with a discussion of limitations and prospects for the conjugate AO technique in two-photon biological microscopy. PMID:26367938

Scatter sensitive microscopic techniques to identify contrasting mucosal structures in ultrahigh-resolution optical coherence tomograms of mouse colon

NASA Astrophysics Data System (ADS)

Tumlinson, Alexandre R.; Hariri, Lida P.; Drexler, Wolfgang; Barton, Jennifer K.

2008-02-01

Optical coherence tomography, optical coherence microscopy, reflectance confocal microscopy, and darkfield microscopy all derive contrast from the intensity of endogenous tissue scatter. We have imaged excised mouse colon tissue with these complimentary technologies to make conclusions about structural origins of scatter in the mouse colonic mucosa observed with endoscopic OCT. We find hyperintense scattering both from the cytoplasm of epithelial cells and from the boundary between epithelia and the lamina propria. We find almost no scatter from the portion of epithelial cells containing the nucleus. These observations substantiate explanations for the appearance of colonic crypts and the luminal surface.

Single-shot optical sectioning using two-color probes in HiLo fluorescence microscopy.

PubMed

Muro, Eleonora; Vermeulen, Pierre; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit; Fragola, Alexandra; Loriette, Vincent

2011-06-08

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Axial range of conjugate adaptive optics in two-photon microscopy.

PubMed

Paudel, Hari P; Taranto, John; Mertz, Jerome; Bifano, Thomas

2015-08-10

We describe an adaptive optics technique for two-photon microscopy in which the deformable mirror used for aberration compensation is positioned in a plane conjugate to the plane of the aberration. We demonstrate in a proof-of-principle experiment that this technique yields a large field of view advantage in comparison to standard pupil-conjugate adaptive optics. Further, we show that the extended field of view in conjugate AO is maintained over a relatively large axial translation of the deformable mirror with respect to the conjugate plane. We conclude with a discussion of limitations and prospects for the conjugate AO technique in two-photon biological microscopy.

Investigation of the spatial distribution of second-order nonlinearity in thermally poled optical fibers.

PubMed

An, Honglin; Fleming, Simon

2005-05-02

The spatial distribution of second-order nonlinearity in thermally poled optical fibers was characterized by second-harmonic microscopy. The second-order nonlinearity was found to be confined to a thin layer close to the anode surface and progressed further into the silica as the poling time increased. Position uncertainty of the anode metal wire was observed to have an effect, as the nonlinear layers were found not always symmetrically located around the nearest points between the anode and cathode. Optical microscopy results were obtained on etched poled fiber cross-sections and compared with those from second-harmonic microscopy.

Tutorial on photoacoustic tomography

NASA Astrophysics Data System (ADS)

Zhou, Yong; Yao, Junjie; Wang, Lihong V.

2016-06-01

Photoacoustic tomography (PAT) has become one of the fastest growing fields in biomedical optics. Unlike pure optical imaging, such as confocal microscopy and two-photon microscopy, PAT employs acoustic detection to image optical absorption contrast with high-resolution deep into scattering tissue. So far, PAT has been widely used for multiscale anatomical, functional, and molecular imaging of biological tissues. We focus on PAT's basic principles, major implementations, imaging contrasts, and recent applications.

Double-clad photonic crystal fiber coupler for compact nonlinear optical microscopy imaging.

PubMed

Fu, Ling; Gu, Min

2006-05-15

A 1 x 2 double-clad photonic crystal fiber coupler is fabricated by the fused tapered method, showing a low excess loss of 1.1 dB and a splitting ratio of 97/3 over the entire visible and near-infrared wavelength range. In addition to the property of splitting the laser power, the double-clad feature of the coupler facilitates the separation of a near-infrared single-mode beam from a visible multimode beam, which is ideal for nonlinear optical microscopy imaging. In conjunction with a gradient-index lens, this coupler is used to construct a miniaturized microscope based on two-photon fluorescence and second-harmonic generation. Three-dimensional nonlinear optical images demonstrate potential applications of the coupler to compact all-fiber and nonlinear optical microscopy and endoscopy.

Photon gating in four-dimensional ultrafast electron microscopy.

PubMed

Hassan, Mohammed T; Liu, Haihua; Baskin, John Spencer; Zewail, Ahmed H

2015-10-20

Ultrafast electron microscopy (UEM) is a pivotal tool for imaging of nanoscale structural dynamics with subparticle resolution on the time scale of atomic motion. Photon-induced near-field electron microscopy (PINEM), a key UEM technique, involves the detection of electrons that have gained energy from a femtosecond optical pulse via photon-electron coupling on nanostructures. PINEM has been applied in various fields of study, from materials science to biological imaging, exploiting the unique spatial, energy, and temporal characteristics of the PINEM electrons gained by interaction with a "single" light pulse. The further potential of photon-gated PINEM electrons in probing ultrafast dynamics of matter and the optical gating of electrons by invoking a "second" optical pulse has previously been proposed and examined theoretically in our group. Here, we experimentally demonstrate this photon-gating technique, and, through diffraction, visualize the phase transition dynamics in vanadium dioxide nanoparticles. With optical gating of PINEM electrons, imaging temporal resolution was improved by a factor of 3 or better, being limited only by the optical pulse widths. This work enables the combination of the high spatial resolution of electron microscopy and the ultrafast temporal response of the optical pulses, which provides a promising approach to attain the resolution of few femtoseconds and attoseconds in UEM.

Photon gating in four-dimensional ultrafast electron microscopy

PubMed Central

Hassan, Mohammed T.; Liu, Haihua; Baskin, John Spencer; Zewail, Ahmed H.

2015-01-01

Ultrafast electron microscopy (UEM) is a pivotal tool for imaging of nanoscale structural dynamics with subparticle resolution on the time scale of atomic motion. Photon-induced near-field electron microscopy (PINEM), a key UEM technique, involves the detection of electrons that have gained energy from a femtosecond optical pulse via photon–electron coupling on nanostructures. PINEM has been applied in various fields of study, from materials science to biological imaging, exploiting the unique spatial, energy, and temporal characteristics of the PINEM electrons gained by interaction with a “single” light pulse. The further potential of photon-gated PINEM electrons in probing ultrafast dynamics of matter and the optical gating of electrons by invoking a “second” optical pulse has previously been proposed and examined theoretically in our group. Here, we experimentally demonstrate this photon-gating technique, and, through diffraction, visualize the phase transition dynamics in vanadium dioxide nanoparticles. With optical gating of PINEM electrons, imaging temporal resolution was improved by a factor of 3 or better, being limited only by the optical pulse widths. This work enables the combination of the high spatial resolution of electron microscopy and the ultrafast temporal response of the optical pulses, which provides a promising approach to attain the resolution of few femtoseconds and attoseconds in UEM. PMID:26438835

Multimodal hyperspectral optical microscopy

DOE PAGES

Novikova, Irina V.; Smallwood, Chuck R.; Gong, Yu; ...

2017-09-02

We describe a unique and convenient approach to multimodal hyperspectral optical microscopy, herein achieved by coupling a portable and transferable hyperspectral imager to various optical microscopes. The experimental and data analysis schemes involved in recording spectrally and spatially resolved fluorescence, dark field, and optical absorption micrographs are illustrated through prototypical measurements targeting selected model systems. Namely, hyperspectral fluorescence micrographs of isolated fluorescent beads are employed to ensure spectral calibration of our detector and to gauge the attainable spatial resolution of our measurements; the recorded images are diffraction-limited. Moreover, spatially over-sampled absorption spectroscopy of a single lipid (18:1 Liss Rhod PE)more » layer reveals that optical densities on the order of 10-3 may be resolved by spatially averaging the recorded optical signatures. We also briefly illustrate two applications of our setup in the general areas of plasmonics and cell biology. Most notably, we deploy hyperspectral optical absorption microscopy to identify and image algal pigments within a single live Tisochrysis lutea cell. Overall, this work paves the way for multimodal multidimensional spectral imaging measurements spanning the realms of several scientific disciples.« less

Multimodal hyperspectral optical microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Novikova, Irina V.; Smallwood, Chuck R.; Gong, Yu

We describe a unique and convenient approach to multimodal hyperspectral optical microscopy, herein achieved by coupling a portable and transferable hyperspectral imager to various optical microscopes. The experimental and data analysis schemes involved in recording spectrally and spatially resolved fluorescence, dark field, and optical absorption micrographs are illustrated through prototypical measurements targeting selected model systems. Namely, hyperspectral fluorescence micrographs of isolated fluorescent beads are employed to ensure spectral calibration of our detector and to gauge the attainable spatial resolution of our measurements; the recorded images are diffraction-limited. Moreover, spatially over-sampled absorption spectroscopy of a single lipid (18:1 Liss Rhod PE)more » layer reveals that optical densities on the order of 10-3 may be resolved by spatially averaging the recorded optical signatures. We also briefly illustrate two applications of our setup in the general areas of plasmonics and cell biology. Most notably, we deploy hyperspectral optical absorption microscopy to identify and image algal pigments within a single live Tisochrysis lutea cell. Overall, this work paves the way for multimodal multidimensional spectral imaging measurements spanning the realms of several scientific disciples.« less

Functional photoacoustic microscopy of pH

NASA Astrophysics Data System (ADS)

Chatni, M. Rameez; Yao, Junjie; Danielli, Amos; Favazza, Christopher P.; Maslov, Konstantin I.; Wang, Lihong V.

2012-02-01

pH is a tightly regulated indicator of metabolic activity. In mammalian systems, imbalance of pH regulation may result from or result in serious illness. Even though the regulation system of pH is very robust, tissue pH can be altered in many diseases such as cancer, osteoporosis and diabetes mellitus. Traditional high-resolution optical imaging techniques, such as confocal microscopy, routinely image pH in cells and tissues using pH sensitive fluorescent dyes, which change their fluorescence properties with the surrounding pH. Since strong optical scattering in biological tissue blurs images at greater depths, high-resolution pH imaging is limited to penetration depths of 1mm. Here, we report photoacoustic microscopy (PAM) of commercially available pH-sensitive fluorescent dye in tissue phantoms. Using both opticalresolution photoacoustic microscopy (OR-PAM), and acoustic resolution photoacoustic microscopy (AR-PAM), we explored the possibility of recovering the pH values in tissue phantoms. In this paper, we demonstrate that PAM was capable of recovering pH values up to a depth of 2 mm, greater than possible with other forms of optical microscopy.

The study of documentary photographs of the early 20th century by the optical coherence microscopy method

NASA Astrophysics Data System (ADS)

Ryseva, Ekaterina; Zhukova, Ekaterina

2013-05-01

The wide field and spectral methods of optical coherence microscopy were used for extensive studying the photographs printed in the early 20th century. Tomographic images (B-scans) of photo and paper materials are presented and discussed.

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,…

Application of scanning acoustic microscopy to advanced structural ceramics

NASA Technical Reports Server (NTRS)

Vary, Alex; Klima, Stanley J.

1987-01-01

A review is presentod of research investigations of several acoustic microscopy techniques for application to structural ceramics for advanced heat engines. Results obtained with scanning acoustic microscopy (SAM), scanning laser acoustic microscopy (SLAM), scanning electron acoustic microscopy (SEAM), and photoacoustic microscopy (PAM) are compared. The techniques were evaluated on research samples of green and sintered monolithic silicon nitrides and silicon carbides in the form of modulus-of-rupture bars containing deliberately introduced flaws. Strengths and limitations of the techniques are described with emphasis on statistics of detectability of flaws that constitute potential fracture origins.

Advanced optical manufacturing digital integrated system

NASA Astrophysics Data System (ADS)

Tao, Yizheng; Li, Xinglan; Li, Wei; Tang, Dingyong

2012-10-01

It is necessarily to adapt development of advanced optical manufacturing technology with modern science technology development. To solved these problems which low of ration, ratio of finished product, repetition, consistent in big size and high precision in advanced optical component manufacturing. Applied business driven and method of Rational Unified Process, this paper has researched advanced optical manufacturing process flow, requirement of Advanced Optical Manufacturing integrated System, and put forward architecture and key technology of it. Designed Optical component core and Manufacturing process driven of Advanced Optical Manufacturing Digital Integrated System. the result displayed effective well, realized dynamic planning Manufacturing process, information integration improved ratio of production manufactory.

SEM/EDS and optical microscopy analyses of microplastics in ocean trawl and fish guts.

PubMed

Wang, Zhong-Min; Wagner, Jeff; Ghosal, Sutapa; Bedi, Gagandeep; Wall, Stephen

2017-12-15

Microplastic particles from Atlantic and Pacific Ocean trawls, lab-fed fish guts and ocean fish guts have been characterized using optical microscopy and SEM/EDS in terms of size, morphology, and chemistry. We assessed whether these measurements could serve as a rapid screening process for subsequent identification of the likely microplastic candidates by micro-spectroscopy. Optical microscopy enabled morphological classification of the types of particles or fibers present in the sample, as well as the quantification of particle size ranges and fiber lengths. SEM/EDS analysis was used to rule out non-plastic particles and screen the prepared samples for potential microplastic, based on their element signatures and surface characteristics. Chlorinated plastics such as polyvinyl chloride (PVC) could be easily identified with SEM/EDS due to their unique elemental signatures including chlorine, as could mineral species that are falsely identified as plastics by optical microscopy. Particle morphology determined by optical microscopy and SEM suggests the fish ingested particles contained both degradation fragments from larger plastic pieces and also manufactured microplastics. SEM images of microplastic particle surfaces revealed characteristic cracks consistent with environmental exposure, as well as pigment particles consistent with manufactured materials. Most of the microplastic surfaces in the fish guts and ocean trawls were covered with biofilms, radiolarians, and crustaceans. Many of the fish stomachs contained micro-shell pieces which visually resembled microplastics. Copyright © 2017 Elsevier B.V. All rights reserved.

Dimensional metrology of lab-on-a-chip internal structures: a comparison of optical coherence tomography with confocal fluorescence microscopy.

PubMed

Reyes, D R; Halter, M; Hwang, J

2015-07-01

The characterization of internal structures in a polymeric microfluidic device, especially of a final product, will require a different set of optical metrology tools than those traditionally used for microelectronic devices. We demonstrate that optical coherence tomography (OCT) imaging is a promising technique to characterize the internal structures of poly(methyl methacrylate) devices where the subsurface structures often cannot be imaged by conventional wide field optical microscopy. The structural details of channels in the devices were imaged with OCT and analyzed with an in-house written ImageJ macro in an effort to identify the structural details of the channel. The dimensional values obtained with OCT were compared with laser-scanning confocal microscopy images of channels filled with a fluorophore solution. Attempts were also made using confocal reflectance and interferometry microscopy to measure the channel dimensions, but artefacts present in the images precluded quantitative analysis. OCT provided the most accurate estimates for the channel height based on an analysis of optical micrographs obtained after destructively slicing the channel with a microtome. OCT may be a promising technique for the future of three-dimensional metrology of critical internal structures in lab-on-a-chip devices because scans can be performed rapidly and noninvasively prior to their use. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Ion polished Cr/Sc attosecond multilayer mirrors for high water window reflectivity

DOE PAGES

Guggenmos, Alexander; Radünz, Stefan; Rauhut, Roman; ...

2014-01-20

Recent advances in the development of attosecond soft X-ray sources ranging into the water window spectral range, between the 1s states of carbon and oxygen (284 eV–543 eV), are also driving the development of suited broadband multilayer optics for steering and shaping attosecond pulses. The relatively low intensity of current High Harmonic Generation (HHG) soft X-ray sources calls for an efficient use of photons, thus the development of low-loss multilayer optics is of uttermost importance. Here, we report about the realization of broadband Cr/Sc attosecond multilayer mirrors with nearly atomically smooth interfaces by an optimized ion beam deposition and assistedmore » interface polishing process. This yields to our knowledge highest multilayer mirror reflectivity at 300 eV near normal incidence. The results are verified by transmission electron microscopy (TEM) and soft/hard X-ray reflectometry.« less

Ion polished Cr/Sc attosecond multilayer mirrors for high water window reflectivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guggenmos, Alexander; Radünz, Stefan; Rauhut, Roman

Recent advances in the development of attosecond soft X-ray sources ranging into the water window spectral range, between the 1s states of carbon and oxygen (284 eV–543 eV), are also driving the development of suited broadband multilayer optics for steering and shaping attosecond pulses. The relatively low intensity of current High Harmonic Generation (HHG) soft X-ray sources calls for an efficient use of photons, thus the development of low-loss multilayer optics is of uttermost importance. Here, we report about the realization of broadband Cr/Sc attosecond multilayer mirrors with nearly atomically smooth interfaces by an optimized ion beam deposition and assistedmore » interface polishing process. This yields to our knowledge highest multilayer mirror reflectivity at 300 eV near normal incidence. The results are verified by transmission electron microscopy (TEM) and soft/hard X-ray reflectometry.« less

Sub-50 nm metrology on extreme ultra violet chemically amplified resist—A systematic assessment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maas, D. J., E-mail: diederik.maas@tno.nl; Herfst, R.; Veldhoven, E. van

2015-10-15

With lithographic patterning dimensions decreasing well below 50 nm, it is of high importance to understand metrology at such small scales. This paper presents results obtained from dense arrays of contact holes (CHs) with various Critical Dimension (CD) between 15 and 50 nm, as patterned in a chemically amplified resist using an ASML EUV scanner and measured at ASML and TNO. To determine the differences between various (local) CD metrology techniques, we conducted an experiment using optical scatterometry, CD-Scanning Electron Microscopy (CD-SEM), Helium ion Microscopy (HIM), and Atomic Force Microscopy (AFM). CD-SEM requires advanced beam scan strategies to mitigate samplemore » charging; the other tools did not need that. We discuss the observed main similarities and differences between the various techniques. To this end, we assessed the spatial frequency content in the raw images for SEM, HIM, and AFM. HIM and AFM resolve the highest spatial frequencies, which are attributed to the more localized probe-sample interaction for these techniques. Furthermore, the SEM, HIM, and AFM waveforms are analyzed in detail. All techniques show good mutual correlation, albeit the reported CD values systematically differ significantly. HIM systematically reports a 25% higher CD uniformity number than CD-SEM for the same arrays of CHs, probably because HIM has a higher resolution than the CD-SEM used in this assessment. A significant speed boost for HIM and AFM is required before these techniques are to serve the demanding industrial metrology applications like optical critical dimension and CD-SEM do nowadays.« less

Non-invasive in vivo characterization of skin wound healing using label-free multiphoton microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Jones, Jake D.; Majid, Fariah; Ramser, Hallie; Quinn, Kyle P.

2017-02-01

Non-healing ulcerative wounds, such as diabetic foot ulcers, are challenging to diagnose and treat due to their numerous possible etiologies and the variable efficacy of advanced wound care products. Thus, there is a critical need to develop new quantitative biomarkers and diagnostic technologies that are sensitive to wound status in order to guide care. The objective of this study was to evaluate the utility of label-free multiphoton microscopy for characterizing wound healing dynamics in vivo and identifying potential differences in diabetic wounds. We isolated and measured an optical redox ratio of FAD/(NADH+FAD) autofluorescence to provide three-dimensional maps of local cellular metabolism. Using a mouse model of wound healing, in vivo imaging at the wound edge identified a significant decrease in the optical redox ratio of the epidermis (p≤0.0103) between Days 3 through 14 compared to Day 1. This decrease in redox ratio coincided with a decrease in NADH fluorescence lifetime and thickening of the epithelium, collectively suggesting a sensitivity to keratinocyte hyperproliferation. In contrast to normal wounds, we have found that keratinocytes from diabetic wounds remain in a proliferative state at later time points with a lower redox ratio at the wound edge. Microstructural organization and composition was also measured from second harmonic generation imaging of collagen and revealed differences between diabetic and non-diabetic wounds. Our work demonstrates label-free multiphoton microscopy offers potential to provide non-invasive structural and functional biomarkers associated with different stages of skin wound healing, which may be used to detect delayed healing and guide treatment.

Sub-50 nm metrology on extreme ultra violet chemically amplified resist—A systematic assessment

NASA Astrophysics Data System (ADS)

Maas, D. J.; Fliervoet, T.; Herfst, R.; van Veldhoven, E.; Meessen, J.; Vaenkatesan, V.; Sadeghian, H.

2015-10-01

With lithographic patterning dimensions decreasing well below 50 nm, it is of high importance to understand metrology at such small scales. This paper presents results obtained from dense arrays of contact holes (CHs) with various Critical Dimension (CD) between 15 and 50 nm, as patterned in a chemically amplified resist using an ASML EUV scanner and measured at ASML and TNO. To determine the differences between various (local) CD metrology techniques, we conducted an experiment using optical scatterometry, CD-Scanning Electron Microscopy (CD-SEM), Helium ion Microscopy (HIM), and Atomic Force Microscopy (AFM). CD-SEM requires advanced beam scan strategies to mitigate sample charging; the other tools did not need that. We discuss the observed main similarities and differences between the various techniques. To this end, we assessed the spatial frequency content in the raw images for SEM, HIM, and AFM. HIM and AFM resolve the highest spatial frequencies, which are attributed to the more localized probe-sample interaction for these techniques. Furthermore, the SEM, HIM, and AFM waveforms are analyzed in detail. All techniques show good mutual correlation, albeit the reported CD values systematically differ significantly. HIM systematically reports a 25% higher CD uniformity number than CD-SEM for the same arrays of CHs, probably because HIM has a higher resolution than the CD-SEM used in this assessment. A significant speed boost for HIM and AFM is required before these techniques are to serve the demanding industrial metrology applications like optical critical dimension and CD-SEM do nowadays.

HelioScan: a software framework for controlling in vivo microscopy setups with high hardware flexibility, functional diversity and extendibility.

PubMed

Langer, Dominik; van 't Hoff, Marcel; Keller, Andreas J; Nagaraja, Chetan; Pfäffli, Oliver A; Göldi, Maurice; Kasper, Hansjörg; Helmchen, Fritjof

2013-04-30

Intravital microscopy such as in vivo imaging of brain dynamics is often performed with custom-built microscope setups controlled by custom-written software to meet specific requirements. Continuous technological advancement in the field has created a need for new control software that is flexible enough to support the biological researcher with innovative imaging techniques and provide the developer with a solid platform for quickly and easily implementing new extensions. Here, we introduce HelioScan, a software package written in LabVIEW, as a platform serving this dual role. HelioScan is designed as a collection of components that can be flexibly assembled into microscope control software tailored to the particular hardware and functionality requirements. Moreover, HelioScan provides a software framework, within which new functionality can be implemented in a quick and structured manner. A specific HelioScan application assembles at run-time from individual software components, based on user-definable configuration files. Due to its component-based architecture, HelioScan can exploit synergies of multiple developers working in parallel on different components in a community effort. We exemplify the capabilities and versatility of HelioScan by demonstrating several in vivo brain imaging modes, including camera-based intrinsic optical signal imaging for functional mapping of cortical areas, standard two-photon laser-scanning microscopy using galvanometric mirrors, and high-speed in vivo two-photon calcium imaging using either acousto-optic deflectors or a resonant scanner. We recommend HelioScan as a convenient software framework for the in vivo imaging community. Copyright © 2013 Elsevier B.V. All rights reserved.

Miniaturized video-microscopy system for near real-time water quality biomonitoring using microfluidic chip-based devices

NASA Astrophysics Data System (ADS)

Huang, Yushi; Nigam, Abhimanyu; Campana, Olivia; Nugegoda, Dayanthi; Wlodkowic, Donald

2016-12-01

Biomonitoring studies apply biological responses of sensitive biomonitor organisms to rapidly detect adverse environmental changes such as presence of physic-chemical stressors and toxins. Behavioral responses such as changes in swimming patterns of small aquatic invertebrates are emerging as sensitive endpoints to monitor aquatic pollution. Although behavioral responses do not deliver information on an exact type or the intensity of toxicants present in water samples, they could provide orders of magnitude higher sensitivity than lethal endpoints such as mortality. Despite the advantages of behavioral biotests performed on sentinel organisms, their wider application in real-time and near realtime biomonitoring of water quality is limited by the lack of dedicated and automated video-microscopy systems. Current behavioral analysis systems rely mostly on static test conditions and manual procedures that are time-consuming and labor intensive. Tracking and precise quantification of locomotory activities of multiple small aquatic organisms requires high-resolution optical data recording. This is often problematic due to small size of fast moving animals and limitations of culture vessels that are not specially designed for video data recording. In this work, we capitalized on recent advances in miniaturized CMOS cameras, high resolution optics and biomicrofluidic technologies to develop near real-time water quality sensing using locomotory activities of small marine invertebrates. We present proof-of-concept integration of high-resolution time-resolved video recording system and high-throughput miniaturized perfusion biomicrofluidic platform for optical tracking of nauplii of marine crustacean Artemia franciscana. Preliminary data demonstrate that Artemia sp. exhibits rapid alterations of swimming patterns in response to toxicant exposure. The combination of video-microscopy and biomicrofluidic platform facilitated straightforward recording of fast moving objects. We envisage that prospectively such system can be scaled up to perform high-throughput water quality sensing in a robotic biomonitoring facility.

DMD-based quantitative phase microscopy and optical diffraction tomography

NASA Astrophysics Data System (ADS)

Zhou, Renjie

2018-02-01

Digital micromirror devices (DMDs), which offer high speed and high degree of freedoms in steering light illuminations, have been increasingly applied to optical microscopy systems in recent years. Lately, we introduced DMDs into digital holography to enable new imaging modalities and break existing imaging limitations. In this paper, we will first present our progress in using DMDs for demonstrating laser-illumination Fourier ptychographic microscopy (FPM) with shotnoise limited detection. After that, we will present a novel common-path quantitative phase microscopy (QPM) system based on using a DMD. Building on those early developments, a DMD-based high speed optical diffraction tomography (ODT) system has been recently demonstrated, and the results will also be presented. This ODT system is able to achieve video-rate 3D refractive-index imaging, which can potentially enable observations of high-speed 3D sample structural changes.

Optical contrast and refractive index of natural van der Waals heterostructure nanosheets of franckeite

PubMed Central

Gant, Patricia; Ghasemi, Foad; Maeso, David; Munuera, Carmen; López-Elvira, Elena; Frisenda, Riccardo; De Lara, David Pérez; Rubio-Bollinger, Gabino; Garcia-Hernandez, Mar

2017-01-01

We study mechanically exfoliated nanosheets of franckeite by quantitative optical microscopy. The analysis of transmission-mode and epi-illumination-mode optical microscopy images provides a rapid method to estimate the thickness of the exfoliated flakes at first glance. A quantitative analysis of the optical contrast spectra by means of micro-reflectance allows one to determine the refractive index of franckeite over a broad range of the visible spectrum through a fit of the acquired spectra to a model based on the Fresnel law. PMID:29181292

Bio-optic signatures for advanced glycation end products in the skin in streptozotocin (STZ) Induced Diabetes (Conference Presentation)

NASA Astrophysics Data System (ADS)

Saidian, Mayer; Ponticorvo, Adrien; Rowland, Rebecca A.; Balbado, Melisa L.; Lentsch, Griffin; Balu, Mihaela; Alexander, Micheal; Shiri, Li; Lakey, Jonathan R. T.; Durkin, Anthony J.; Kohen, Roni; Tromberg, Bruce J.

2017-02-01

Type 1diabetes (T1D) is an autoimmune disorder that occurs due to the rapid destruction of insulin-producing beta cells, leading to insulin deficiency and the inability to regulate blood glucose levels and leads to destructive secondary complications. Advanced glycation end (AGEs) products, the result of the cross-linking of reducing sugars and proteins within the tissues, are one of the key causes of major complications associated with diabetes such as renal failure, blindness, nerve damage and vascular changes. Non-invasive techniques to detect AGEs are important for preventing the harmful effects of AGEs during diabetes mellitus. In this study, we utilized multiphoton microscopy to image biopsies taken from control rats and compared them to biopsies taken from streptozotocin (STZ) induced adult male diabetic rats. This was done at two and four weeks after the induction of hyperglycemia (>400 mg/dL) specifically to evaluate the effects of glycation on collagen. We chose to use an in-situ multiphoton microscopy method that combines multiphoton auto-florescence (AF) and second harmonic generation (SHG) to detect the microscopic influence of glycation. Initial results show high auto-florescence levels were present on the collagen, as a result of the accumulation of AGEs only two weeks after the STZ injection and considerably higher levels were present four weeks after the STZ injection. Future projects could involve evaluating advanced glycation end products in a clinical trial of diabetic patients.

Tutorial on photoacoustic tomography

PubMed Central

Zhou, Yong; Yao, Junjie; Wang, Lihong V.

2016-01-01

Abstract. Photoacoustic tomography (PAT) has become one of the fastest growing fields in biomedical optics. Unlike pure optical imaging, such as confocal microscopy and two-photon microscopy, PAT employs acoustic detection to image optical absorption contrast with high-resolution deep into scattering tissue. So far, PAT has been widely used for multiscale anatomical, functional, and molecular imaging of biological tissues. We focus on PAT’s basic principles, major implementations, imaging contrasts, and recent applications. PMID:27086868

Microstructure Evolution and Mechanical Behavior of 2219 Aluminum Alloys Additively Fabricated by the Cold Metal Transfer Process

PubMed Central

Fang, Xuewei; Li, Hui; Li, Chaolong; Lu, Bingheng

2018-01-01

In this research, four different welding arc modes including conventional cold metal transfer (CMT), CMT-Pulse (CMT-P), CMT-Advanced (CMT-ADV), and CMT pulse advanced (CMT-PADV) were used to deposit 2219-Al wire. The effects of different arc modes on porosity, pore size distribution, microstructure evolution, and mechanical properties were thoroughly investigated. The statistical analysis of the porosity and its size distribution indicated that the CMT-PADV process gave the smallest pore area percentage and pore aspect ratio, and had almost no larger pores. The results from optical microscopy, scanning electron microscopy, and fractographic morphology proved that uniform and fine equiaxed grains, evenly distributed Al2Cu second phase particles were formed during the CMT-PADV process. Furthermore, the X-ray diffraction test ascertained that the CMT-PADV sample had the smallest lattice parameter and the highest solute Cu content. Besides, the tensile strength could reach 283 MPa, the data scattering was the smallest, and the strength scattering of the sample in the horizontal direction was the shortest. In addition, the strength properties were nearly isotropic, with only 5 MPa difference in the vertical and horizontal directions. The above mentioned results indicated that the mechanical properties of 2219 aluminum alloy was improved using the CMT-PADV arc mode. PMID:29772708

Microstructure Evolution and Mechanical Behavior of 2219 Aluminum Alloys Additively Fabricated by the Cold Metal Transfer Process.

PubMed

Fang, Xuewei; Zhang, Lijuan; Li, Hui; Li, Chaolong; Huang, Ke; Lu, Bingheng

2018-05-16

In this research, four different welding arc modes including conventional cold metal transfer (CMT), CMT-Pulse (CMT-P), CMT-Advanced (CMT-ADV), and CMT pulse advanced (CMT-PADV) were used to deposit 2219-Al wire. The effects of different arc modes on porosity, pore size distribution, microstructure evolution, and mechanical properties were thoroughly investigated. The statistical analysis of the porosity and its size distribution indicated that the CMT-PADV process gave the smallest pore area percentage and pore aspect ratio, and had almost no larger pores. The results from optical microscopy, scanning electron microscopy, and fractographic morphology proved that uniform and fine equiaxed grains, evenly distributed Al₂Cu second phase particles were formed during the CMT-PADV process. Furthermore, the X-ray diffraction test ascertained that the CMT-PADV sample had the smallest lattice parameter and the highest solute Cu content. Besides, the tensile strength could reach 283 MPa, the data scattering was the smallest, and the strength scattering of the sample in the horizontal direction was the shortest. In addition, the strength properties were nearly isotropic, with only 5 MPa difference in the vertical and horizontal directions. The above mentioned results indicated that the mechanical properties of 2219 aluminum alloy was improved using the CMT-PADV arc mode.

Hyperlens-array-implemented optical microscopy

NASA Astrophysics Data System (ADS)

Iwanaga, Masanobu

2014-08-01

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

Noise characterization of broadband fiber Cherenkov radiation as a visible-wavelength source for optical coherence tomography and two-photon fluorescence microscopy.

PubMed

Tu, Haohua; Zhao, Youbo; Liu, Yuan; Liu, Yuan-Zhi; Boppart, Stephen

2014-08-25

Optical sources in the visible region immediately adjacent to the near-infrared biological optical window are preferred in imaging techniques such as spectroscopic optical coherence tomography of endogenous absorptive molecules and two-photon fluorescence microscopy of intrinsic fluorophores. However, existing sources based on fiber supercontinuum generation are known to have high relative intensity noise and low spectral coherence, which may degrade imaging performance. Here we compare the optical noise and pulse compressibility of three high-power fiber Cherenkov radiation sources developed recently, and evaluate their potential to replace the existing supercontinuum sources in these imaging techniques.

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.

Going "open" with mesoscopy: a new dimension on multi-view imaging.

PubMed

Gualda, Emilio; Moreno, Nuno; Tomancak, Pavel; Martins, Gabriel G

2014-03-01

OpenSPIM and OpenSpinMicroscopy emerged as open access platforms for Light Sheet and Optical Projection Imaging, often called as optical mesoscopy techniques. Both projects can be easily reproduced using comprehensive online instructions that should foster the implementation and further development of optical imaging techniques with sample rotation control. This additional dimension in an open system offers the possibility to make multi-view microscopy easily modified and will complement the emerging commercial solutions. Furthermore, it is deeply based on other open platforms such as MicroManager and Arduino, enabling development of tailored setups for very specific biological questions. In our perspective, the open access principle of OpenSPIM and OpenSpinMicroscopy is a game-changer, helping the concepts of light sheet and optical projection tomography (OPT) to enter the mainstream of biological imaging.

A Next-Generation Hard X-Ray Nanoprobe Beamline for In Situ Studies of Energy Materials and Devices

NASA Astrophysics Data System (ADS)

Maser, Jörg; Lai, Barry; Buonassisi, Tonio; Cai, Zhonghou; Chen, Si; Finney, Lydia; Gleber, Sophie-Charlotte; Jacobsen, Chris; Preissner, Curt; Roehrig, Chris; Rose, Volker; Shu, Deming; Vine, David; Vogt, Stefan

2014-01-01

The Advanced Photon Source is developing a suite of new X-ray beamlines to study materials and devices across many length scales and under real conditions. One of the flagship beamlines of the APS upgrade is the In Situ Nanoprobe (ISN) beamline, which will provide in situ and operando characterization of advanced energy materials and devices under varying temperatures, gas ambients, and applied fields, at previously unavailable spatial resolution and throughput. Examples of materials systems include inorganic and organic photovoltaic systems, advanced battery systems, fuel cell components, nanoelectronic devices, advanced building materials and other scientifically and technologically relevant systems. To characterize these systems at very high spatial resolution and trace sensitivity, the ISN will use both nanofocusing mirrors and diffractive optics to achieve spots sizes as small as 20 nm. Nanofocusing mirrors in Kirkpatrick-Baez geometry will provide several orders of magnitude increase in photon flux at a spatial resolution of 50 nm. Diffractive optics such as zone plates and/or multilayer Laue lenses will provide a highest spatial resolution of 20 nm. Coherent diffraction methods will be used to study even small specimen features with sub-10 nm relevant length scale. A high-throughput data acquisition system will be employed to significantly increase operations efficiency and usability of the instrument. The ISN will provide full spectroscopy capabilities to study the chemical state of most materials in the periodic table, and enable X-ray fluorescence tomography. In situ electrical characterization will enable operando studies of energy and electronic devices such as photovoltaic systems and batteries. We describe the optical concept for the ISN beamline, the technical design, and the approach for enabling a broad variety of in situ studies. We furthermore discuss the application of hard X-ray microscopy to study defects in multi-crystalline solar cells, one of the lines of inquiries for which the ISN is being developed.

Chapter 7: Total internal reflection fluorescence microscopy.

PubMed

Axelrod, Daniel

2008-01-01

Total internal reflection fluorescence microscopy (TIRFM), also known as evanescent wave microscopy, is used in a wide range of applications, particularly to view single molecules attached to planar surfaces and to study the position and dynamics of molecules and organelles in living culture cells near the contact regions with the glass coverslip. TIRFM selectively illuminates fluorophores only in a very thin (less than 100 nm deep) layer near the substrate, thereby avoiding excitation of fluorophores outside this subresolution optical section. This chapter reviews the history, current applications in cell biology and biochemistry, basic optical theory, combinations with numerous other optical and spectroscopic approaches, and a range of setup methods, both commercial and custom.

Probing Subdiffraction Limit Separations with Plasmon Coupling Microscopy: Concepts and Applications

PubMed Central

Wu, Linxi

2014-01-01

Due to their advantageous materials properties, noble metal nanoparticles are versatile tools in biosensing and imaging. A characteristic feature of gold and silver nanoparticles is their ability to sustain localized surface plasmons that provide both large optical cross-sections and extraordinary photophysical stability. Plasmon Coupling Microscopy takes advantage of the beneficial optical properties and utilizes electromagnetic near-field coupling between individual noble metal nanoparticle labels to resolve subdiffraction limit separations in an all-optical fashion. This Tutorial provides an introduction into the physical concepts underlying distance dependent plasmon coupling, discusses potential experimental implementations of Plasmon Coupling Microscopy, and reviews applications in the area of biosensing and imaging. PMID:24390574

Noninvasive label-free monitoring of cosmetics and pharmaceuticals in human skin using nonlinear optical microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Osseiran, Sam; Wang, Hequn; Evans, Conor L.

2017-02-01

Over the past decade, nonlinear optical microscopy has seen a dramatic rise in its use in research settings due to its noninvasiveness, enhanced penetration depth, intrinsic optical sectioning, and the ability to probe chemical compounds with molecular specificity without exogenous contrast agents. Nonlinear optical techniques including two-photon excitation fluorescence (2PEF), fluorescence lifetime imaging microscopy (FLIM), second harmonic generation (SHG), coherent anti-Stokes and stimulated Raman scattering (CARS and SRS, respectively), as well as transient and sum frequency absorption (TA and SFA, respectively), have been widely used to explore the physiology and microanatomy of skin. Recently, these modalities have shed light on dermal processes that could not have otherwise been observed, including the spatiotemporal monitoring of cosmetics and pharmaceuticals. However, a challenge quickly arises when studying such chemicals in a dermatological context: many exogenous compounds have optical signatures that can interfere with the signals that would otherwise be acquired from intact skin. For example, oily solvents exhibit strong signals when probing CH2 vibrations with CARS/SRS; chemical sun filters appear bright in 2PEF microscopy; and darkly colored compounds readily absorb light across a broad spectrum, producing strong TA/SFA signals. Thus, this discussion will first focus on the molecular contrast in skin that can be probed using the aforementioned nonlinear optical techniques. This will be followed by an overview of strategies that take advantage of the exogenous compounds' optical signatures to probe spatiotemporal dynamics while preserving endogenous information from skin.

Deep-tissue two-photon imaging in brain and peripheral nerve with a compact high-pulse energy ytterbium fiber laser

NASA Astrophysics Data System (ADS)

Fontaine, Arjun K.; Kirchner, Matthew S.; Caldwell, John H.; Weir, Richard F.; Gibson, Emily A.

2018-02-01

Two-photon microscopy is a powerful tool of current scientific research, allowing optical visualization of structures below the surface of tissues. This is of particular value in neuroscience, where optically accessing regions within the brain is critical for the continued advancement in understanding of neural circuits. However, two-photon imaging at significant depths have typically used Ti:Sapphire based amplifiers that are prohibitively expensive and bulky. In this study, we demonstrate deep tissue two-photon imaging using a compact, inexpensive, turnkey operated Ytterbium fiber laser (Y-Fi, KM Labs). The laser is based on all-normal dispersion (ANDi) that provides short pulse durations and high pulse energies. Depth measurements obtained in ex vivo mouse cortex exceed those obtainable with standard two-photon microscopes using Ti:Sapphire lasers. In addition to demonstrating the capability of deep-tissue imaging in the brain, we investigated imaging depth in highly-scattering white matter with measurements in sciatic nerve showing limited optical penetration of heavily myelinated nerve tissue relative to grey matter.

Leakage radiation interference microscopy.

PubMed

Descrovi, Emiliano; Barakat, Elsie; Angelini, Angelo; Munzert, Peter; De Leo, Natascia; Boarino, Luca; Giorgis, Fabrizio; Herzig, Hans Peter

2013-09-01

We present a proof of principle for a new imaging technique combining leakage radiation microscopy with high-resolution interference microscopy. By using oil immersion optics it is demonstrated that amplitude and phase can be retrieved from optical fields, which are evanescent in air. This technique is illustratively applied for mapping a surface mode propagating onto a planar dielectric multilayer on a thin glass substrate. The surface mode propagation constant estimated after Fourier transformation of the measured complex field is well matched with an independent measurement based on back focal plane imaging.

Advances in Bio-Optical Imaging for the Diagnosis of Early Oral Cancer

PubMed Central

Olivo, Malini; Bhuvaneswari, Ramaswamy; Keogh, Ivan

2011-01-01

Oral cancer is among the most common malignancies worldwide, therefore early detection and treatment is imperative. The 5-year survival rate has remained at a dismal 50% for the past several decades. The main reason for the poor survival rate is the fact that most of the oral cancers, despite the general accessibility of the oral cavity, are not diagnosed until the advanced stage. Early detection of the oral tumors and its precursor lesions may be the most effective means to improve clinical outcome and cure most patients. One of the emerging technologies is the use of non-invasive in vivo tissue imaging to capture the molecular changes at high-resolution to improve the detection capability of early stage disease. This review will discuss the use of optical probes and highlight the role of optical imaging such as autofluorescence, fluorescence diagnosis (FD), laser confocal endomicroscopy (LCE), surface enhanced Raman spectroscopy (SERS), optical coherence tomography (OCT) and confocal reflectance microscopy (CRM) in early oral cancer detection. FD is a promising method to differentiate cancerous lesions from benign, thus helping in the determination of adequate resolution of surgical resection margin. LCE offers in vivo cellular imaging of tissue structures from surface to subsurface layers and has demonstrated the potential to be used as a minimally invasive optical biopsy technique for early diagnosis of oral cancer lesions. SERS was able to differentiate between normal and oral cancer patients based on the spectra acquired from saliva of patients. OCT has been used to visualize the detailed histological features of the oral lesions with an imaging depth down to 2–3 mm. CRM is an optical tool to noninvasively image tissue with near histological resolution. These comprehensive diagnostic modalities can also be used to define surgical margin and to provide a direct assessment of the therapeutic effectiveness. PMID:24310585

[Gene sequencing by scanning molecular exciton microscopy]. Progress report, October 1, 1990--September 30, 1991

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1991-12-31

This report details progress made in setting up a laboratory for optical microscopy of genes. The apparatus including a fluorescence microscope, a scanning optical microscope, various spectrometers, and supporting computers is described. Results in developing photon and exciton tips, and in preparing samples are presented. (GHH)

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

NASA Astrophysics Data System (ADS)

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

2009-11-01

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

Aberration control in 4Pi nanoscopy: definitions, properties, and applications (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hao, Xiang; Allgeyer, Edward S.; Velasco, Mary Grace M.; Booth, Martin J.; Bewersdorf, Joerg

2016-03-01

The development of fluorescence microscopy, which allows live-cell imaging with high labeling specificity, has made the visualization of cellular architecture routine. However, for centuries, the spatial resolution of optical microscopy was fundamentally limited by diffraction. The past two decades have seen a revolution in far-field optical nanoscopy (or "super-resolution" microscopy). The best 3D resolution is achieved by optical nanoscopes like the isoSTED or the iPALM/4Pi-SMS, which utilize two opposing objective lenses in a coherent manner. These system are, however, also more complex and the required interference conditions demand precise aberration control. Our research involves developing novel adaptive optics techniques that enable high spatial and temporal resolution imaging for biological applications. In this talk, we will discuss how adaptive optics can enhance dual-objective lens nanoscopes. We will demonstrate how adaptive optics devices provide unprecedented freedom to manipulate the light field in isoSTED nanoscopy, allow to realize automatic beam alignment, suppress the inherent side-lobes of the point-spread function, and dynamically compensate for sample-induced aberrations. We will present both the theoretical groundwork and the experimental confirmations.

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.

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.

Wavefront correction using machine learning methods for single molecule localization microscopy

NASA Astrophysics Data System (ADS)

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

2015-03-01

Optical Aberrations are a major challenge in imaging biological samples. In particular, in single molecule localization (SML) microscopy techniques (STORM, PALM, etc.) a high Strehl ratio point spread function (PSF) is necessary to achieve sub-diffraction resolution. Distortions in the PSF shape directly reduce the resolution of SML microscopy. The system aberrations caused by the imperfections in the optics and instruments can be compensated using Adaptive Optics (AO) techniques prior to imaging. However, aberrations caused by the biological sample, both static and dynamic, have to be dealt with in real time. A challenge for wavefront correction in SML microscopy is a robust optimization approach in the presence of noise because of the naturally high fluctuations in photon emission from single molecules. Here we demonstrate particle swarm optimization for real time correction of the wavefront using an intensity independent metric. We show that the particle swarm algorithm converges faster than the genetic algorithm for bright fluorophores.

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

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

PubMed

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

2016-09-01

Optoacoustic (OA) microscopy using an all-optical system based on the probe beam deflection technique (PBDT) for detection of laser-induced acoustic signals was investigated as an alternative to conventional piezoelectric transducers. PBDT provides a number of advantages for OA microscopy including (i) efficient coupling of laser excitation energy to the samples being imaged through the probing laser beam, (ii) undistorted coupling of acoustic waves to the detector without the need for separation of the optical and acoustic paths, (iii) high sensitivity and (iv) ultrawide bandwidth. Because of the unimpeded optical path in PBDT, diffraction-limited lateral resolution can be readily achieved. The sensitivity of the current PBDT sensor of 22 μV/Pa and its noise equivalent pressure (NEP) of 11.4 Pa are comparable with these parameters of the optical micro-ring resonator and commercial piezoelectric ultrasonic transducers. Benefits of the present prototype OA microscope were demonstrated by successfully resolving micron-size details in histological sections of cardiac muscle.

Light Sheet Fluorescence Microscopy (LSFM)

PubMed Central

Adams, Michael W.; Loftus, Andrew F.; Dunn, Sarah E.; Joens, Matthew S.; Fitzpatrick, James A.J.

2015-01-01

The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light Sheet Fluorescent Microscopy (LSFM), a century old idea (Siedentopf and Zsigmondy, 1902) made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light sheet based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements. PMID:25559221

Multiphoton imaging of myogenic differentiation in gelatin-based hydrogels as tissue engineering scaffolds.

PubMed

Kim, Min Jeong; Shin, Yong Cheol; Lee, Jong Ho; Jun, Seung Won; Kim, Chang-Seok; Lee, Yunki; Park, Jong-Chul; Lee, Soo-Hong; Park, Ki Dong; Han, Dong-Wook

2016-01-01

Hydrogels can serve as three-dimensional (3D) scaffolds for cell culture and be readily injected into the body. Recent advances in the image technology for 3D scaffolds like hydrogels have attracted considerable attention to overcome the drawbacks of ordinary imaging technologies such as optical and fluorescence microscopy. Multiphoton microscopy (MPM) is an effective method based on the excitation of two-photons. In the present study, C2C12 myoblasts differentiated in 3D gelatin hydroxyphenylpropionic acid (GHPA) hydrogels were imaged by using a custom-built multiphoton excitation fluorescence microscopy to compare the difference in the imaging capacity between conventional microscopy and MPM. The physicochemical properties of GHPA hydrogels were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. In addition, the cell viability and proliferation of C2C12 myoblasts cultured in the GHPA hydrogels were analyzed by using Live/Dead Cell and CCK-8 assays, respectively. It was found that C2C12 cells were well grown and normally proliferated in the hydrogels. Furthermore, the hydrogels were shown to be suitable to facilitate the myogenic differentiation of C2C12 cells incubated in differentiation media, which had been corroborated by MPM. It was very hard to get clear images from a fluorescence microscope. Our findings suggest that the gelatin-based hydrogels can be beneficially utilized as 3D scaffolds for skeletal muscle engineering and that MPM can be effectively applied to imaging technology for tissue regeneration.

76 FR 12144 - Advanced Optics Electronics, Inc.; Order of Suspension of Trading

Federal Register 2010, 2011, 2012, 2013, 2014

2011-03-04

... SECURITIES AND EXCHANGE COMMISSION [File No. 500-1] Advanced Optics Electronics, Inc.; Order of... lack of current and accurate information concerning the securities of Advanced Optics Electronics, Inc... in Advanced Optics Electronics, Inc. Therefore, it is ordered, pursuant to Section 12(k) of the...

Quantitative photoacoustic microscopy of optical absorption coefficients from acoustic spectra in the optical diffusive regime

NASA Astrophysics Data System (ADS)

Guo, Zijian; Favazza, Christopher; Garcia-Uribe, Alejandro; Wang, Lihong V.

2012-06-01

Photoacoustic (PA) microscopy (PAM) can image optical absorption contrast with ultrasonic spatial resolution in the optical diffusive regime. Conventionally, accurate quantification in PAM requires knowledge of the optical fluence attenuation, acoustic pressure attenuation, and detection bandwidth. We circumvent this requirement by quantifying the optical absorption coefficients from the acoustic spectra of PA signals acquired at multiple optical wavelengths. With the acoustic spectral method, the absorption coefficients of an oxygenated bovine blood phantom at 560, 565, 570, and 575 nm were quantified with errors of <3%. We also quantified the total hemoglobin concentration and hemoglobin oxygen saturation in a live mouse. Compared with the conventional amplitude method, the acoustic spectral method provides greater quantification accuracy in the optical diffusive regime. The limitations of the acoustic spectral method was also discussed.

Quantitative photoacoustic microscopy of optical absorption coefficients from acoustic spectra in the optical diffusive regime

PubMed Central

Guo, Zijian; Favazza, Christopher; Garcia-Uribe, Alejandro

2012-01-01

Abstract. Photoacoustic (PA) microscopy (PAM) can image optical absorption contrast with ultrasonic spatial resolution in the optical diffusive regime. Conventionally, accurate quantification in PAM requires knowledge of the optical fluence attenuation, acoustic pressure attenuation, and detection bandwidth. We circumvent this requirement by quantifying the optical absorption coefficients from the acoustic spectra of PA signals acquired at multiple optical wavelengths. With the acoustic spectral method, the absorption coefficients of an oxygenated bovine blood phantom at 560, 565, 570, and 575 nm were quantified with errors of <3%. We also quantified the total hemoglobin concentration and hemoglobin oxygen saturation in a live mouse. Compared with the conventional amplitude method, the acoustic spectral method provides greater quantification accuracy in the optical diffusive regime. The limitations of the acoustic spectral method was also discussed. PMID:22734767

Quantitative photoacoustic microscopy of optical absorption coefficients from acoustic spectra in the optical diffusive regime.

PubMed

Guo, Zijian; Favazza, Christopher; Garcia-Uribe, Alejandro; Wang, Lihong V

2012-06-01

Photoacoustic (PA) microscopy (PAM) can image optical absorption contrast with ultrasonic spatial resolution in the optical diffusive regime. Conventionally, accurate quantification in PAM requires knowledge of the optical fluence attenuation, acoustic pressure attenuation, and detection bandwidth. We circumvent this requirement by quantifying the optical absorption coefficients from the acoustic spectra of PA signals acquired at multiple optical wavelengths. With the acoustic spectral method, the absorption coefficients of an oxygenated bovine blood phantom at 560, 565, 570, and 575 nm were quantified with errors of <3%. We also quantified the total hemoglobin concentration and hemoglobin oxygen saturation in a live mouse. Compared with the conventional amplitude method, the acoustic spectral method provides greater quantification accuracy in the optical diffusive regime. The limitations of the acoustic spectral method was also discussed.

Localization microscopy of DNA in situ using Vybrant{sup ®} DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Żurek-Biesiada, Dominika; Szczurek, Aleksander T.; Prakash, Kirti

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant{sup ®} DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei ofmore » fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10{sup 6} signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100 nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy. - Highlights: • Super-resolution imaging of nuclear DNA with Vybrant Violet and blue excitation. • 90nm resolution images of DNA structures in optically thick eukaryotic nuclei. • Enhanced resolution confirms the existence of DNA-free regions inside the nucleus. • Optimized imaging conditions enable multicolor super-resolution imaging.« less

Laser beam shaping for biomedical microscopy techniques

NASA Astrophysics Data System (ADS)

Laskin, Alexander; Kaiser, Peter; Laskin, Vadim; Ostrun, Aleksei

2016-04-01

Uniform illumination of a working field is very important in optical systems of confocal microscopy and various implementations of fluorescence microscopy like TIR, SSIM, STORM, PALM to enhance performance of these laser-based research techniques. Widely used TEM00 laser sources are characterized by essentially non-uniform Gaussian intensity profile which leads usually to non-uniform intensity distribution in a microscope working field or in a field of microlenses array of a confocal microscope optical system, this non-uniform illumination results in instability of measuring procedure and reducing precision of quantitative measurements. Therefore transformation of typical Gaussian distribution of a TEM00 laser to flat-top (top hat) profile is an actual technical task, it is solved by applying beam shaping optics. Due to high demands to optical image quality the mentioned techniques have specific requirements to a uniform laser beam: flatness of phase front and extended depth of field, - from this point of view the microscopy techniques are similar to holography and interferometry. There are different refractive and diffractive beam shaping approaches used in laser industrial and scientific applications, but only few of them are capable to fulfil the optimum conditions for beam quality required in discussed microscopy techniques. We suggest applying refractive field mapping beam shapers πShaper, which operational principle presumes almost lossless transformation of Gaussian to flat-top beam with flatness of output wavefront, conserving of beam consistency, providing collimated low divergent output beam, high transmittance, extended depth of field, negligible wave aberration, and achromatic design provides capability to work with several lasers with different wavelengths simultaneously. The main function of a beam shaper is transformation of laser intensity profile, further beam transformation to provide optimum for a particular technique spot size and shape has to be realized by an imaging optical system which can include microscope objectives and tube lenses. This paper will describe design basics of refractive beam shapers and optical layouts of their applying in microscopy systems. Examples of real implementations and experimental results will be presented as well.

Nonlinear Optical Spectroscopy of Two-Dimensional Materials

NASA Astrophysics Data System (ADS)

Cui, Qiannan

Nonlinear optical properties of two-dimensional (2D) materials, such as transition metal dichalcogenides (TMDs), graphene, black phosphorus, and so on, play a key role of understanding nanoscale light-matter interactions, as well as developing nanophotonics applications from solar cells to quantum computation. With ultrafast lasers, we experimentally study nonlinear optical properties of 2D materials. Employing transient absorption microscopy, we study several members of 2D materials, such as WSe2, TiS3 and ReS2. The dynamical saturable absorption process of 2D excitons is spatiotemporally resolved. Intrinsic parameters of these 2D materials, such as exciton lifetime, exciton diffusion coefficient, and exciton mobility, are effectively measured. Especially, in-plane anisotropy of transient absorption and diffusive transport is observed for 2D excitons in monolayer ReS2, demonstrating the in-plane degree of freedom. Furthermore, with quantum interference and control nanoscopy, we all-optically inject, detect and manipulate nanoscale ballistic charge currents in a ReS2 thin film. By tuning the phase difference between one photon absorption and two photon absorption transition paths, sub-picosecond timescale of ballistic currents is coherently controlled for the first time in TMDs. In addition, the spatial resolution is two-order of magnitude smaller than optical diffraction limit. The second-order optical nonlinearity of 2D monolayers is resolved by second harmonic generation (SHG) microscopy. We measure the second-order susceptibility of monolayer MoS 2. The angular dependence of SHG in monolayer MoS2 shows strong symmetry dependence on its crystal lattice structure. Hence, second harmonic generation microscopy can serve as a powerful tool to noninvasively determine the crystalline directions of 2D monolayers. The real and imaginary parts of third-order optical nonlinearity of 2D monolayers are resolved by third harmonic generation (THG) microscopy and two-photon transient absorption microscopy, respectively. With third harmonic generation microscopy, we observe strong and anisotropic THG in monolayer and multilayer ReS2. Comparing with 2D materials with hexagonal lattice, such as MoS2, the third-order susceptibility is higher by one order of magnitude in ReS2 with a distorted 1T structure. The in-plane anisotropy of THG is attributed to the lattice distortion in ReS2 after comparing with a symmetry analysis. With two-photon transient absorption microscopy, we observe a giant two-photon absorption coefficient of monolayer WS2.

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.

Advances in high-resolution imaging--techniques for three-dimensional imaging of cellular structures.

PubMed

Lidke, Diane S; Lidke, Keith A

2012-06-01

A fundamental goal in biology is to determine how cellular organization is coupled to function. To achieve this goal, a better understanding of organelle composition and structure is needed. Although visualization of cellular organelles using fluorescence or electron microscopy (EM) has become a common tool for the cell biologist, recent advances are providing a clearer picture of the cell than ever before. In particular, advanced light-microscopy techniques are achieving resolutions below the diffraction limit and EM tomography provides high-resolution three-dimensional (3D) images of cellular structures. The ability to perform both fluorescence and electron microscopy on the same sample (correlative light and electron microscopy, CLEM) makes it possible to identify where a fluorescently labeled protein is located with respect to organelle structures visualized by EM. Here, we review the current state of the art in 3D biological imaging techniques with a focus on recent advances in electron microscopy and fluorescence super-resolution techniques.

Grid-enhanced X-ray coded aperture microscopy with polycapillary optics

PubMed Central

Sowa, Katarzyna M.; Last, Arndt; Korecki, Paweł

2017-01-01

Polycapillary devices focus X-rays by means of multiple reflections of X-rays in arrays of bent glass capillaries. The size of the focal spot (typically 10–100 μm) limits the resolution of scanning, absorption and phase-contrast X-ray imaging using these devices. At the expense of a moderate resolution, polycapillary elements provide high intensity and are frequently used for X-ray micro-imaging with both synchrotrons and X-ray tubes. Recent studies have shown that the internal microstructure of such an optics can be used as a coded aperture that encodes high-resolution information about objects located inside the focal spot. However, further improvements to this variant of X-ray microscopy will require the challenging fabrication of tailored devices with a well-defined capillary microstructure. Here, we show that submicron coded aperture microscopy can be realized using a periodic grid that is placed at the output surface of a polycapillary optics. Grid-enhanced X-ray coded aperture microscopy with polycapillary optics does not rely on the specific microstructure of the optics but rather takes advantage only of its focusing properties. Hence, submicron X-ray imaging can be realized with standard polycapillary devices and existing set-ups for micro X-ray fluorescence spectroscopy. PMID:28322316

Grid-enhanced X-ray coded aperture microscopy with polycapillary optics.

PubMed

Sowa, Katarzyna M; Last, Arndt; Korecki, Paweł

2017-03-21

Polycapillary devices focus X-rays by means of multiple reflections of X-rays in arrays of bent glass capillaries. The size of the focal spot (typically 10-100 μm) limits the resolution of scanning, absorption and phase-contrast X-ray imaging using these devices. At the expense of a moderate resolution, polycapillary elements provide high intensity and are frequently used for X-ray micro-imaging with both synchrotrons and X-ray tubes. Recent studies have shown that the internal microstructure of such an optics can be used as a coded aperture that encodes high-resolution information about objects located inside the focal spot. However, further improvements to this variant of X-ray microscopy will require the challenging fabrication of tailored devices with a well-defined capillary microstructure. Here, we show that submicron coded aperture microscopy can be realized using a periodic grid that is placed at the output surface of a polycapillary optics. Grid-enhanced X-ray coded aperture microscopy with polycapillary optics does not rely on the specific microstructure of the optics but rather takes advantage only of its focusing properties. Hence, submicron X-ray imaging can be realized with standard polycapillary devices and existing set-ups for micro X-ray fluorescence spectroscopy.

Al nanogrid electrode for ultraviolet detectors.

PubMed

Ding, G; Deng, J; Zhou, L; Gan, Q; Hwang, J C M; Dierolf, V; Bartoli, F J; Mazuir, C; Schoenfeld, W V

2011-09-15

Optical properties of Al nanogrids of different pitches and gaps were investigated both theoretically and experimentally. Three-dimensional finite-difference time-domain simulation predicted that surface plasmons at the air/Al interface would enhance ultraviolet transmission through the subwavelength gaps of the nanogrid, making it an effective electrode on GaN-based photodetectors to compensate for the lack of transparent electrode and high p-type doping. The predicted transmission enhancement was verified by confocal scanning optical microscopy performed at 365 nm. The quality of the nanogrids fabricated by electron-beam lithography was verified by near-field scanning optical microscopy and scanning electron microscopy. Based on the results, the pitch and gap of the nanogrids can be optimized for the best trade-off between electrical conductivity and optical transmission at different wavelengths. Based on different cutoff wavelengths, the nanogrids can also double as a filter to render photodetectors solar-blind.

Fabrication of bright and thin Zn₂SiO₄ luminescent film for electron beam excitation-assisted optical microscope.

PubMed

Furukawa, Taichi; Kanamori, Satoshi; Fukuta, Masahiro; Nawa, Yasunori; Kominami, Hiroko; Nakanishi, Yoichiro; Sugita, Atsushi; Inami, Wataru; Kawata, Yoshimasa

2015-07-13

We fabricated a bright and thin Zn₂SiO₄ luminescent film to serve as a nanometric light source for high-spatial-resolution optical microscopy based on electron beam excitation. The Zn₂SiO₄ luminescent thin film was fabricated by annealing a ZnO film on a Si₃N₄ substrate at 1000 °C in N₂. The annealed film emitted bright cathodoluminescence compared with the as-deposited film. The film is promising for nano-imaging with electron beam excitation-assisted optical microscopy. We evaluated the spatial resolution of a microscope developed using this Zn₂SiO₄ luminescent thin film. This is the first report of the investigation and application of ZnO/Si₃N₄ annealed at a high temperature (1000 °C). The fabricated Zn₂SiO₄ film is expected to enable high-frame-rate dynamic observation with ultra-high resolution using our electron beam excitation-assisted optical microscopy.

Electro-optical interfacial effects on a graphene/π-conjugated organic semiconductor hybrid system

PubMed Central

Araujo, Karolline A S; Cury, Luiz A; Matos, Matheus J S; Fernandes, Thales F D; Cançado, Luiz G

2018-01-01

The influence of graphene and retinoic acid (RA) – a π-conjugated organic semiconductor – interface on their hybrid system is investigated. The physical properties of the interface are assessed via scanning probe microscopy, optical spectroscopy (photoluminescence and Raman) and ab initio calculations. The graphene/RA interaction induces the formation of a well-organized π-conjugated self-assembled monolayer (SAM) at the interface. Such structural organization leads to the high optical emission efficiency of the RA SAM, even at room temperature. Additionally, photo-assisted electrical force microscopy, photo-assisted scanning Kelvin probe microscopy and Raman spectroscopy indicate a RA-induced graphene doping and photo-charge generation. Finally, the optical excitation of the RA monolayer generates surface potential changes on the hybrid system. In summary, interface-induced organized structures atop 2D materials may have an important impact on both design and operation of π-conjugated nanomaterial-based hybrid systems. PMID:29600157

Localization microscopy of DNA in situ using Vybrant(®) DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution.

PubMed

Żurek-Biesiada, Dominika; Szczurek, Aleksander T; Prakash, Kirti; Mohana, Giriram K; Lee, Hyun-Keun; Roignant, Jean-Yves; Birk, Udo J; Dobrucki, Jurek W; Cremer, Christoph

2016-05-01

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant(®) DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei of fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10(6) signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy. Copyright © 2016. Published by Elsevier Inc.

Three-dimensional imaging of dislocation propagation during crystal growth and dissolution

PubMed Central

Schenk, Anna S.; Kim, Yi-Yeoun; Kulak, Alexander N.; Campbell, James M.; Nisbet, Gareth; Meldrum, Fiona C.; Robinson, Ian K.

2015-01-01

Atomic level defects such as dislocations play key roles in determining the macroscopic properties of crystalline materials 1,2. Their effects range from increased chemical reactivity 3,4 to enhanced mechanical properties 5,6. Dislocations have been widely studied using traditional techniques such as X-ray diffraction and optical imaging. Recent advances have enabled atomic force microscopy to study single dislocations 7 in two-dimensions (2D), while transmission electron microscopy (TEM) can now visualise strain fields in three-dimensions (3D) with near atomic resolution 8–10. However, these techniques cannot offer 3D imaging of the formation or movement of dislocations during dynamic processes. Here, we describe how Bragg Coherent Diffraction Imaging (BCDI) 11,12 can be used to visualize in 3D, the entire network of dislocations present within an individual calcite crystal during repeated growth and dissolution cycles. These investigations demonstrate the potential of BCDI for studying the mechanisms underlying the response of crystalline materials to external stimuli. PMID:26030304

Imaging the morphological change of tissue structure during the early phase of esophageal tumor progression using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Xu, Jian; Kang, Deyong; Xu, Meifang; Zhu, Xiaoqin; Zhuo, Shuangmu; Chen, Jianxin

2012-12-01

Esophageal cancer is a common malignancy with a very poor prognosis. Successful strategies for primary prevention and early detection are critically needed to control this disease. Multiphoton microscopy (MPM) is becoming a novel optical tool of choice for imaging tissue architecture and cellular morphology by two-photon excited fluorescence. In this study, we used MPM to image microstructure of human normal esophagus, carcinoma in situ (CIS), and early invasive carcinoma in order to establish the morphological features to differentiate these tissues. The diagnostic features such as the appearance of cancerous cells, the significant loss of stroma, the absence of the basement membrane were extracted to distinguish between normal and cancerous esophagus tissue. These results correlated well with the paired histological findings. With the advancement of clinically miniaturized MPM and the multi-photon probe, combining MPM with standard endoscopy will therefore allow us to make a real-time in vivo diagnosis of early esophageal cancer at the cellular level.

Holographic techniques for cellular fluorescence microscopy

NASA Astrophysics Data System (ADS)

Kim, Myung K.

2017-04-01

We have constructed a prototype instrument for holographic fluorescence microscopy (HFM) based on self-interference incoherent digital holography (SIDH) and demonstrate novel imaging capabilities such as differential 3D fluorescence microscopy and optical sectioning by compressive sensing.

Metal-Coated Optical Fibers for High Temperature Applications

NASA Technical Reports Server (NTRS)

Zeakes, Jason; Murphy, Kent; Claus, Richard; Greene, Jonathan; Tran, Tuan

1996-01-01

A DC magnetron sputtering system has been used to actively coat optical fibers with hermetic metal coatings during the fiber draw process. Thin films of Inconel 625 have been deposited on optical fibers and annealed in air at 2000 F. Scanning electron microscopy and Auger electron microscopy have been used to investigate the morphology and composition of the films prior to and following thermal cycling. Issues to be addressed include film adhesion, other coating materials, and a discussion of additional applications for this novel technology.

Two-Photon Fluorescence Microscopy Developed for Microgravity Fluid Physics

NASA Technical Reports Server (NTRS)

Fischer, David G.; Zimmerli, Gregory A.; Asipauskas, Marius

2004-01-01

Recent research efforts within the Microgravity Fluid Physics Branch of the NASA Glenn Research Center have necessitated the development of a microscope capable of high-resolution, three-dimensional imaging of intracellular structure and tissue morphology. Standard optical microscopy works well for thin samples, but it does not allow the imaging of thick samples because of severe degradation caused by out-of-focus object structure. Confocal microscopy, which is a laser-based scanning microscopy, provides improved three-dimensional imaging and true optical sectioning by excluding the out-of-focus light. However, in confocal microscopy, out-of-focus object structure is still illuminated by the incoming beam, which can lead to substantial photo-bleaching. In addition, confocal microscopy is plagued by limited penetration depth, signal loss due to the presence of a confocal pinhole, and the possibility of live-cell damage. Two-photon microscopy is a novel form of laser-based scanning microscopy that allows three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon microscopy, it utilizes the nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption because of the nonlinear (i.e., quadratic) electric field dependence, so an ultrafast pulsed laser source must typically be employed. On the other hand, this stringent energy density requirement effectively localizes fluorophore excitation to the focal volume. Consequently, two-photon microscopy provides optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction in photo-damage because of the longer excitation wavelength, a reduction in background fluorescence, and a 4 increase in penetration depth over confocal methods because of the reduction in Rayleigh scattering.

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

PubMed Central

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

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.

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

NASA Astrophysics Data System (ADS)

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

2011-01-01

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

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

Chemical Silver Coating of Fiber Tips in Near-Field Scanning Optical Microscopy

NASA Technical Reports Server (NTRS)

Vikram, Chandra S.; Witherow, William K.

1998-01-01

We report what is believed to be the first experimental demonstration of silver coating by a wet chemical process on tapered fiber tips used in near-field scanning optical microscopy. The process is at room temperature and pressure and takes only a few minutes to complete. Many tips can be simultaneously coated.

Soil Analysis Micro-Mission Concepts Derived from the MSP 2001 Mars Environmental Compatibility Assessment (MECA)

NASA Technical Reports Server (NTRS)

Hecht, M. H.; Meloy, T. P.; Anderson, M. S.; Buehler, M. G.; Frant, M. A.; Grannan, S. M.; Fuerstenau, S. D.; Keller, H. U.; Markiewicz, W. J.; Marshall, J.

1999-01-01

The Mars Environmental Compatibility Assessment (MECA) will evaluate the Martian environment for soil and dust-related hazards to human exploration as part of the Mars Surveyor Program 2001 Lander. The integrated MECA payload contains a wet-chemistry laboratory, a microscopy station, an electrometer to characterize the electrostatic environment, and arrays of material patches to study abrasion and adhesion. Heritage will be all-important for low cost micro-missions, and adaptations of instruments developed for the Pathfinder, '98 and '01 Landers should be strong contenders for '03 flights. This talk has three objectives: (1) Familiarize the audience with MECA instrument capabilities; (2) present concepts for stand-alone and/or mobile versions of MECA instruments; and (3) broaden the context of the MECA instruments from human exploration to a comprehensive scientific survey of Mars. Due to time limitations, emphasis will be on the chemistry and microscopy experiments. Ion-selective electrodes and related sensors in MECA's wet-chemistry laboratory will evaluate total dissolved solids, redox potential, pH, and the concentration of many soluble ions and gases in wet Martian soil. These electrodes can detect potentially dangerous heavy-metal ions, emitted pathogenic gases, and the soil's corrosive potential, and experiments will include cyclic voltammetry and anodic stripping. For experiments beyond 2001, enhancements could allow multiple use of the cells (for mobile experiments) and reagent addition (for quantitative mineralogical and exobiological analysis). MECA's microscopy station combines optical and atomic-force microscopy (AFM) in an actively focused, controlled illumination environment to image particles from millimeters to nanometers in size. Careful selection of substrates allows controlled experiments in adhesion, abrasion, hardness, aggregation, magnetic and other properties. Special tools allow primitive manipulation (brushing and scraping) of samples. Soil particle properties including size, shape, color, hardness, adhesive potential (electrostatic and magnetic), will be determined using an array of sample receptacles and collection substrates. The simple, rugged atomic-force microscope will image in the submicron size range and has the capability of performing a particle-by-particle analysis of the dust and soil. Future implementations might enhance the optical microscopy with spectroscopy, or incorporate advanced AFM techniques for thermogravimetric and chemical analysis.

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

PubMed Central

Wolenski, Joseph S.; Julich, Doerthe

2014-01-01

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy. PMID:24600334

Plant cell wall characterization using scanning probe microscopy techniques

PubMed Central

Yarbrough, John M; Himmel, Michael E; Ding, Shi-You

2009-01-01

Lignocellulosic biomass is today considered a promising renewable resource for bioenergy production. A combined chemical and biological process is currently under consideration for the conversion of polysaccharides from plant cell wall materials, mainly cellulose and hemicelluloses, to simple sugars that can be fermented to biofuels. Native plant cellulose forms nanometer-scale microfibrils that are embedded in a polymeric network of hemicelluloses, pectins, and lignins; this explains, in part, the recalcitrance of biomass to deconstruction. The chemical and structural characteristics of these plant cell wall constituents remain largely unknown today. Scanning probe microscopy techniques, particularly atomic force microscopy and its application in characterizing plant cell wall structure, are reviewed here. We also further discuss future developments based on scanning probe microscopy techniques that combine linear and nonlinear optical techniques to characterize plant cell wall nanometer-scale structures, specifically apertureless near-field scanning optical microscopy and coherent anti-Stokes Raman scattering microscopy. PMID:19703302

Nanoscale Phase-Separated Structure in Core-Shell Nanoparticles of SiO2-Si1-xGexO2 Glass Revealed by Electron Microscopy.

PubMed

Kubo, Yugo; Yonezawa, Kazuhiro

2017-09-05

SiO 2 -based optical fibers are indispensable components of modern information communication technologies. It has recently become increasingly important to establish a technique for visualizing the nanoscale phase-separated structure inside SiO 2 -GeO 2 glass nanoparticles during the manufacturing of SiO 2 -GeO 2 fibers. This is because the rapidly increasing price of Ge has made it necessary to improve the Ge yield by clarifying the detailed mechanism of Ge diffusion into SiO 2 . However, direct observation of the internal nanostructure of glass particles has been extremely difficult, mainly due to electrostatic charging and the damage induced by electron and X-ray irradiation. In the present study, we used state-of-the-art scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM), and energy dispersive X-ray spectroscopy (EDX) to examine cross-sectional samples of SiO 2 -GeO 2 particles embedded in an epoxy resin, which were fabricated using a broad Ar ion beam and a focused Ga ion beam. These advanced techniques enabled us to observe the internal phase-separated structure of the nanoparticles. We have for the first time clearly determined the SiO 2 -Si 1-x Ge x O 2 core-shell structure of such particles, the element distribution, the degree of crystallinity, and the quantitative chemical composition of microscopic regions, and we discuss the formation mechanism for the observed structure. The proposed imaging protocol is highly promising for studying the internal structure of various core-shell nanoparticles, which affects their catalytic, optical, and electronic properties.

Phase Transformations During Cooling of Automotive Steels

NASA Astrophysics Data System (ADS)

Padgett, Matthew C.

This thesis explores the effect of cooling rate on the microstructure and phases in advanced high strength steels (AHSS). In the manufacturing of automobiles, the primary joining mechanism for steel is resistance spot welding (RSW), a process that produces a high heat input and rapid cooling in the welded metal. The effect of RSW on the microstructure of these material systems is critical to understanding their mechanical properties. A dual phase steel, DP-600, and a transformation induced plasticity bainitic-ferritic steel, TBF-1180, were studied to assess the changes to their microstructure that take place in controlled cooling environments and in uncontrolled cooling environments, i.e. resistance spot welding. Continuous cooling transformation (CCT) diagrams were developed using strip specimens of DP-600 and TBF-1180 to determine the phase transformations that occur as a function of cooling rate. The resulting phases were determined using a thermal-mechanical simulator and dilatometry, combined with light optical microscopy and hardness measurements. The resulting phases were compared with RSW specimens where cooling rate was controlled by varying the welding time for two-plate welds. Comparisons were drawn between experimental welds of DP-600 and simulations performed using a commercial welding software. The type and quantity of phases present after RSW were examined using a variety of techniques, including light optical microscopy using several etchants, hardness measurements, and x-ray diffraction (XRD).

Coherent anti-stokes Raman scattering (CARS) microscopy: a novel technique for imaging the retina.

PubMed

Masihzadeh, Omid; Ammar, David A; Kahook, Malik Y; Lei, Tim C

2013-05-01

To image the cellular and noncellular structures of the retina in an intact mouse eye without the application of exogenous fluorescent labels using noninvasive, nondestructive techniques. Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Cross sectional transverse sections and sequential flat (en face) sagittal sections were collected from a region of sclera approximately midway between the limbus and optic nerve. Imaging proceeded from the surface of the sclera to a depth of ∼60 μm. The fluorescent signal from collagen fibers within the sclera was evident in the TPAF channel; the scleral collagen fibers showed no organization and appeared randomly packed. The sclera contained regions lacking TPAF and CARS fluorescence of ∼3 to 15 μm in diameter that could represent small vessels or scleral fibroblasts. Intense punctate CARS signals from the retinal pigment epithelial layer were of a size and shape of retinyl storage esters. Rod outer segments could be identified by the CARS signal from their lipid-rich plasma membranes. CARS microscopy can be used to image the outer regions of the mammalian retina without the use of a fluorescent dye or exogenously expressed recombinant protein. With technical advancements, CARS/TPAF may represent a new avenue for noninvasively imaging the retina and might complement modalities currently used in clinical practice.

Comparison of divided and full pupil configurations for line-scanning confocal microscopy in human skin and oral mucosa

NASA Astrophysics Data System (ADS)

Larson, Bjorg; Abeytunge, Sanjeewa; Glazowski, Chris; Rajadhyaksha, Milind

2012-02-01

Confocal point-scanning microscopy has been showing promise in the detection, diagnosing and mapping of skin lesions in clinical settings. The noninvasive technique allows provides optical sectioning and cellular resolution for in vivo diagnosis of melanoma and basal cell carcinoma and pre-operative and intra-operative mapping of margins. The imaging has also enabled more accurate "guided" biopsies while minimizing the otherwise large number of "blind" biopsies. Despite these translational advances, however, point-scanning technology remains relatively complex and expensive. Line-scanning technology may offer an alternative approach to accelerate translation to the clinic. Line-scanning, using fewer optical components, inexpensive linear-array detectors and custom electronics, may enable smaller, simpler and lower-cost confocal microscopes. A line is formed using a cylindrical lens and scanned through the back focal plane of the objective with a galvanometric scanner. A linear CCD is used for detection. Two pupil configurations were compared for performance in imaging human tissue. In the full-pupil configuration, illumination and detection is made through the full objective pupil. In the divided pupil approach, half the pupil is illuminated and the other half is used for detection. The divided pupil configuration loses spatial and axial resolution due to a diminished NA, but the sectioning capability and rejection of background is improved. Imaging in skin and oral mucosa illustrate the performance of the two configurations.

Effect of Serum and Insulin Modulation on the Organization and Morphology of Matrix Synthesized by Bovine Corneal Stromal Cells

PubMed Central

Bueno, Ericka M.; Saeidi, Nima; Melotti, Suzanna

2009-01-01

The in vitro production of highly organized collagen fibrils by corneal keratocytes in a three-dimensional scaffold-free culture system presents a unique opportunity for the direct observation of organized matrix formation. The objective of this investigation was to develop such a culture system in a glass substrate (for optical accessibility) and to directly examine the effect of reducing serum and/or increasing insulin on the stratification and secretion of aligned matrix by fourth- to fifth-passage bovine corneal stromal keratocytes. Medium concentrations of 0%, 1%, or 10% fetal bovine serum and 0% or 1% insulin–transferrin–selenium were investigated. High-resolution differential interference contrast microscopy, quick-freeze/deep-etch, and conventional transmission electron microscopy were used to monitor the evolution, morphology, and ultrastructure of the cell–matrix constructs. In a medium containing 1% each of serum and insulin–transferrin–selenium, stromal cells stratified and secreted abundant and locally aligned matrix, generating the thickest cell–matrix constructs (allowing handling with forceps). The results of this study have the potential to significantly advance the field of developmental functional engineering of load-bearing tissues by (i) elucidating cues that modulate in vitro cell secretion of organized matrix and (ii) establishing an optically accessible cell culture system for investigating the mechanism of cell secretion of aligned collagen fibrils. PMID:19480568

Developing single-laser sources for multimodal coherent anti-Stokes Raman scattering microscopy

NASA Astrophysics Data System (ADS)

Pegoraro, Adrian Frank

Coherent anti-Stokes Raman scattering (CARS) microscopy has developed rapidly and is opening the door to new types of experiments. This work describes the development of new laser sources for CARS microscopy and their use for different applications. It is specifically focused on multimodal nonlinear optical microscopy—the simultaneous combination of different imaging techniques. This allows us to address a diverse range of applications, such as the study of biomaterials, fluid inclusions, atherosclerosis, hepatitis C infection in cells, and ice formation in cells. For these applications new laser sources are developed that allow for practical multimodal imaging. For example, it is shown that using a single Ti:sapphire oscillator with a photonic crystal fiber, it is possible to develop a versatile multimodal imaging system using optimally chirped laser pulses. This system can perform simultaneous two photon excited fluorescence, second harmonic generation, and CARS microscopy. The versatility of the system is further demonstrated by showing that it is possible to probe different Raman modes using CARS microscopy simply by changing a time delay between the excitation beams. Using optimally chirped pulses also enables further simplification of the laser system required by using a single fiber laser combined with nonlinear optical fibers to perform effective multimodal imaging. While these sources are useful for practical multimodal imaging, it is believed that for further improvements in CARS microscopy sensitivity, new excitation schemes are necessary. This has led to the design of a new, high power, extended cavity oscillator that should be capable of implementing new excitation schemes for CARS microscopy as well as other techniques. Our interest in multimodal imaging has led us to other areas of research as well. For example, a fiber-coupling scheme for signal collection in the forward direction is demonstrated that allows for fluorescence lifetime imaging without significant temporal distortion. Also highlighted is an imaging artifact that is unique to CARS microscopy that can alter image interpretation, especially when using multimodal imaging. By combining expertise in nonlinear optics, laser development, fiber optics, and microscopy, we have developed systems and techniques that will be of benefit for multimodal CARS microscopy.

Non-interferometric deep optical resolution photoacoustic remote sensing microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

HajiReza, Parsin H.; Bell, Kevan L.; Shi, Wei; Zemp, Roger J.

2017-03-01

A novel all-optical non-contact photoacoustic microscopy system is introduced. The confocal configuration is used to ensure detection of initial pressure shock wave-induced intensity reflections at the subsurface origin where pressures are largest. Phantom studies confirm signal dependence on optical absorption, index-contrast, and excitation fluence. Taking advantage of a focused1310 nm interrogation beam, the penetration depth of the system is improved to 2mm for an optical resolution system. High signal-to-noise ratios (>60dB) with 2.5 cm working distance from the objective lens to the sample is achieved. Real-time in-vivo imaging of microvasculature and melanoma tumors are demonstrated.

Handheld optical-resolution photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Lin, Li; Zhang, Pengfei; Xu, Song; Shi, Junhui; Li, Lei; Yao, Junjie; Wang, Lidai; Zou, Jun; Wang, Lihong V.

2017-04-01

Optical-resolution photoacoustic microscopy (OR-PAM) offers label-free in vivo imaging with high spatial resolution by acoustically detecting optical absorption contrasts via the photoacoustic effect. We developed a compact handheld OR-PAM probe for fast photoacoustic imaging. Different from benchtop microscopes, the handheld probe provides flexibility in imaging various anatomical sites. Resembling a cup in size, the probe uses a two-axis water-immersible microelectromechanical system mirror to scan both the illuminating optical beam and resultant acoustic beam. The system performance was tested in vivo by imaging the capillary bed in a mouse ear and both the capillary bed and a mole on a human volunteer.

Evanescent Waves in High Numerical Aperture Aplanatic Solid Immersion Microscopy: Effects of Forbidden Light on Subsurface Imaging (Open Access, Publisher’s Version)

DTIC Science & Technology

2014-03-24

of the aSIL microscopy for semiconductor failure analysis and is applicable to imaging in quantum optics [18], biophotonics [19] and metrology [20...is usually of interest, the model can be adapted to applications in fields such as quantum optics and biophotonics for which the non-resonant

Emerging optical nanoscopy techniques

PubMed Central

Montgomery, Paul C; Leong-Hoi, Audrey

2015-01-01

To face the challenges of modern health care, new imaging techniques with subcellular resolution or detection over wide fields are required. Far field optical nanoscopy presents many new solutions, providing high resolution or detection at high speed. We present a new classification scheme to help appreciate the growing number of optical nanoscopy techniques. We underline an important distinction between superresolution techniques that provide improved resolving power and nanodetection techniques for characterizing unresolved nanostructures. Some of the emerging techniques within these two categories are highlighted with applications in biophysics and medicine. Recent techniques employing wider angle imaging by digital holography and scattering lens microscopy allow superresolution to be achieved for subcellular and even in vivo, imaging without labeling. Nanodetection techniques are divided into four subcategories using contrast, phase, deconvolution, and nanomarkers. Contrast enhancement is illustrated by means of a polarized light-based technique and with strobed phase-contrast microscopy to reveal nanostructures. Very high sensitivity phase measurement using interference microscopy is shown to provide nanometric surface roughness measurement or to reveal internal nanometric structures. Finally, the use of nanomarkers is illustrated with stochastic fluorescence microscopy for mapping intracellular structures. We also present some of the future perspectives of optical nanoscopy. PMID:26491270

Multiple speckle illumination for optical-resolution photoacoustic imaging

NASA Astrophysics Data System (ADS)

Poisson, Florian; Stasio, Nicolino; Moser, Christophe; Psaltis, Demetri; Bossy, Emmanuel

2017-03-01

Optical-resolution photoacoustic microscopy offers exquisite and specific contrast to optical absorption. Conventional approaches generally involves raster scanning a focused spot over the sample. Here, we demonstrate that a full-field illumination approach with multiple speckle illumination can also provide diffraction-limited optical-resolution photoacoustic images. Two different proof-of-concepts are demonstrated with micro-structured test samples. The first approach follows the principle of correlation/ghost imaging,1, 2 and is based on cross-correlating photoacoustic signals under multiple speckle illumination with known speckle patterns measured during a calibration step. The second approach is a speckle scanning microscopy technique, which adapts the technique proposed in fluorescence microscopy by Bertolotti and al.:3 in our work, spatially unresolved photoacoustic measurements are performed for various translations of unknown speckle patterns. A phase-retrieval algorithm is used to reconstruct the object from the knowledge of the modulus of its Fourier Transform yielded by the measurements. Because speckle patterns naturally appear in many various situations, including propagation through biological tissue or multi-mode fibers (for which focusing light is either very demanding if not impossible), speckle-illumination-based photoacoustic microscopy provides a powerful framework for the development of novel reconstruction approaches, well-suited to compressed sensing approaches.2

Tip/tilt-compensated through-focus scanning optical microscopy

NASA Astrophysics Data System (ADS)

Lee, Jun Ho; Park, Jun Hyung; Jeong, Dohwan; Shin, Eun Ji; Park, Chris

2016-11-01

Through-Focus Optical Microscopy (TSOM), with nanometer scale lateral and vertical sensitivity matching those of scanning electron microscopy, has been demonstrated to be utilized for 3D inspection and metrology. There have been sensitivity and instability issues in acquiring through-focus images because TSOM 3D information is indirectly extracted by differentiating a target TSOM image from reference TSOM images. This paper first reports on the optical axis instability that occurs during the scanning process of TSOM when implemented in an existing patterned wafer inspection tool by moving the wafer plane; this is followed by quantitative confirmation of the optical/mechanical instability using a new TSOM tool on an optical bench with a Shack-Hartmann wavefront sensor and a tip/tilt sensor. Then, this paper proposes two tip/tilt compensated TSOM optical acquisition methods that can be applied with adaptive optics. The first method simply adopts a tip/tilt mirror with a quad cell in a simple closed loop, while the second method adopts a highorder deformable mirror with a Shack-Hartmann sensor. The second method is able to correct high-order residual aberrations as well as to perform through-focus scanning without z-axis movement, while the first method is easier to implement in pre-existing wafer inspection systems with only minor modification.

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

PubMed Central

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

2012-01-01

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

Flow Cytometry with Gold Nanoparticles and their Clusters as scattering Contrast Agents: FDTD Simulation of Light-Cell Interaction

PubMed Central

Tanev, Stoyan; Sun, Wenbo; Pond, James; Tuchin, Valery V.; Zharov, Vladimir P.

2010-01-01

The formulation of the Finite-Difference Time-Domain (FDTD) approach is presented in the framework of its potential applications to in vivo flow cytometry based on light scattering. The consideration is focused on comparison of light scattering by a single biological cell alone in controlled refractive index matching conditions and by cells labeled by gold nanoparticles. The optical schematics including phase contrast (OPCM) microscopy as a prospective modality for in vivo flow cytometry is also analyzed. The validation of the FDTD approach for the simulation of flow cytometry may open a new avenue in the development of advanced cytometric techniques based on scattering effects from nanoscale targets. PMID:19670359

Efficient modeling of Bragg coherent x-ray nanobeam diffraction

DOE PAGES

Hruszkewycz, S. O.; Holt, M. V.; Allain, M.; ...

2015-07-02

X-ray Bragg diffraction experiments that utilize tightly focused coherent beams produce complicated Bragg diffraction patterns that depend on scattering geometry, characteristics of the sample, and properties of the x-ray focusing optic. In this paper, we use a Fourier-transform-based method of modeling the 2D intensity distribution of a Bragg peak and apply it to the case of thin films illuminated with a Fresnel zone plate in three different Bragg scattering geometries. Finally, the calculations agree well with experimental coherent diffraction patterns, demonstrating that nanodiffraction patterns can be modeled at nonsymmetric Bragg conditions with this approach—a capability critical for advancing nanofocused x-raymore » diffraction microscopy.« less

Electronic and Optical Properties of Borophene, a Two-dimensional Transparent Metal.

NASA Astrophysics Data System (ADS)

Adamska, Lyudmyla; Sadasivam, Sridhar; Darancet, Pierre; Sharifzadeh, Sahar

Borophene is a recently synthesized metallic sheet that displays many similarities to graphene and has been predicted to be complimentary to graphene as a high density of states, optically transparent 2D conductor. The atomic arrangement of boron in the monolayer strongly depends on the growth substrate and significantly alters the optoelectronic properties. Here, we report a first-principles density functional theory and many-body perturbation theory study aimed at understanding the optoelectronic properties of two likely allotropes of monolayer boron that are consistent with experimental scanning tunneling microscopy images. We predict that despite both systems are metallic, the two allotropes have substantially different bandstructure and optical properties, with one structure being transparent up to 3 eV and the second weakly absorbing in the UV/Vis region. We demonstrate that this strong structure-dependence of optoelectronic properties is present with the application of strain. Lastly, we discuss the strength of electron-phonon and electron-hole interactions within these materials. Overall, we determine that precise control of the growth conditions in necessary for controlled optical properties. This research used resources of the Argonne Leadership Computing Facility, which is a DOE Office of Science User Facility supported under Contract DE-AC02-06CH11357, and the Texas Advanced Computing Center (TACC) at The University of Texas at Austin.

Correlative imaging of biological tissues with apertureless scanning near-field optical microscopy and confocal laser scanning microscopy

PubMed Central

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

2017-01-01

Apertureless scanning near-field optical microscopy (ASNOM) has attracted considerable interest over the past years as a result of its valuable contrast mechanisms and capabilities for optical resolutions in the nanoscale range. However, at this moment the intersections between ASNOM and the realm of bioimaging are scarce, mainly due to data interpretation difficulties linked to the limited body of work performed so far in this field and hence the reduced volume of supporting information. We propose an imaging approach that holds significant potential for alleviating this issue, consisting of correlative imaging of biological specimens using a multimodal system that incorporates ASNOM and confocal laser scanning microscopy (CLSM), which allows placing near-field data into a well understood context of anatomical relevance. We demonstrate this approach on zebrafish retinal tissue. The proposed method holds important implications for the in-depth understanding of biological items through the prism of ASNOM and CLSM data complementarity. PMID:29296474

Damage mechanisms of MoN/SiN multilayer optics for next-generation pulsed XUV light sources.

PubMed

Sobierajski, R; Bruijn, S; Khorsand, A R; Louis, E; van de Kruijs, R W E; Burian, T; Chalupsky, J; Cihelka, J; Gleeson, A; Grzonka, J; Gullikson, E M; Hajkova, V; Hau-Riege, S; Juha, L; Jurek, M; Klinger, D; Krzywinski, J; London, R; Pelka, J B; Płociński, T; Rasiński, M; Tiedtke, K; Toleikis, S; Vysin, L; Wabnitz, H; Bijkerk, F

2011-01-03

We investigated the damage mechanism of MoN/SiN multilayer XUV optics under two extreme conditions: thermal annealing and irradiation with single shot intense XUV pulses from the free-electron laser facility in Hamburg - FLASH. The damage was studied "post-mortem" by means of X-ray diffraction, interference-polarizing optical microscopy, atomic force microscopy, and scanning transmission electron microscopy. Although the timescale of the damage processes and the damage threshold temperatures were different (in the case of annealing it was the dissociation temperature of Mo2N and in the case of XUV irradiation it was the melting temperature of MoN) the main damage mechanism is very similar: molecular dissociation and the formation of N2, leading to bubbles inside the multilayer structure.

Multimodal nonlinear imaging of arabidopsis thaliana root cell

NASA Astrophysics Data System (ADS)

Jang, Bumjoon; Lee, Sung-Ho; Woo, Sooah; Park, Jong-Hyun; Lee, Myeong Min; Park, Seung-Han

2017-07-01

Nonlinear optical microscopy has enabled the possibility to explore inside the living organisms. It utilizes ultrashort laser pulse with long wavelength (greater than 800nm). Ultrashort pulse produces high peak power to induce nonlinear optical phenomenon such as two-photon excitation fluorescence (TPEF) and harmonic generations in the medium while maintaining relatively low average energy pre area. In plant developmental biology, confocal microscopy is widely used in plant cell imaging after the development of biological fluorescence labels in mid-1990s. However, fluorescence labeling itself affects the sample and the sample deviates from intact condition especially when labelling the entire cell. In this work, we report the dynamic images of Arabidopsis thaliana root cells. This demonstrates the multimodal nonlinear optical microscopy is an effective tool for long-term plant cell imaging.

Fabrication and characterization of optical-fiber nanoprobes for scanning near-field optical microscopy.

PubMed

Essaidi, N; Chen, Y; Kottler, V; Cambril, E; Mayeux, C; Ronarch, N; Vieu, C

1998-02-01

The current scanning near-field optical microscopy has been developed with optical-fiber probes obtained by use of either laser-heated pulling or chemical etching. For high-resolution near-field imaging, the detected signal is rapidly attenuated as the aperture size of the probe decreases. It is thus important to fabricate probes optimized for both spot size and optical transmission. We present a two-step fabrication that allowed us to achieve an improved performance of the optical-fiber probes. Initially, a CO(2) laser-heated pulling was used to produce a parabolic transitional taper ending with a top thin filament. Then, a rapid chemical etching with 50% buffered hydrofluoric acid was used to remove the thin filament and to result in a final conical tip on the top of the parabolic transitional taper. Systematically, we obtained optical-fiber nanoprobes with the apex size as small as 10 nm and the final cone angle varying from 15 degrees to 80 degrees . It was found that the optical transmission efficiency increases rapidly as the taper angle increases from 15 degrees to 50 degrees , but a further increase in the taper angle gives rise to important broadening of the spot size. Finally, the fabricated nanoprobes were used in photon-scanning tunneling microscopy, which allowed observation of etched double lines and grating structures with periods as small as 200 nm.

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. (c) 2006 Wiley Periodicals, Inc.

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

Surface Characterization.

ERIC Educational Resources Information Center

Fulghum, J. E.; And Others

1989-01-01

This review is divided into the following analytical methods: ion spectroscopy, electron spectroscopy, scanning tunneling microscopy, atomic force microscopy, optical spectroscopy, desorption techniques, and X-ray techniques. (MVL)

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

PubMed

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

2013-09-09

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.

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.

Giant optical field enhancement in multi-dielectric stacks by photon scanning tunneling microscopy

NASA Astrophysics Data System (ADS)

Ndiaye, C.; Zerrad, M.; Lereu, A. L.; Roche, R.; Dumas, Ph.; Lemarchand, F.; Amra, C.

2013-09-01

Dielectric optical thin films, as opposed to metallic, have been very sparsely explored as good candidates for absorption-based optical field enhancement. In such materials, the low imaginary part of the refractive index implies that absorption processes are usually not predominant. This leads to dielectric-based optical resonances mainly via waveguiding modes. We show here that when properly designed, a multi-layered dielectric thin films stack can give rise to optical resonances linked to total absorption. We report here, on such dielectric stack designed to possess a theoretical optical field enhancement above 1000. Using photon scanning tunneling microscopy, we experimentally evaluate the resulting field enhancement of the stack as well as the associated penetration depth. We thus demonstrate the capability of multi-dielectric stacks in generating giant optical field with tunable penetration depth (down to few dozens of nm).

Holographic Photolysis for Multiple Cell Stimulation in Mouse Hippocampal Slices

PubMed Central

Papagiakoumou, Eirini; Ventalon, Cathie; Angulo, María Cecilia; Emiliani, Valentina

2010-01-01

Background Advanced light microscopy offers sensitive and non-invasive means to image neural activity and to control signaling with photolysable molecules and, recently, light-gated channels. These approaches require precise and yet flexible light excitation patterns. For synchronous stimulation of subsets of cells, they also require large excitation areas with millisecond and micrometric resolution. We have recently developed a new method for such optical control using a phase holographic modulation of optical wave-fronts, which minimizes power loss, enables rapid switching between excitation patterns, and allows a true 3D sculpting of the excitation volumes. In previous studies we have used holographic photololysis to control glutamate uncaging on single neuronal cells. Here, we extend the use of holographic photolysis for the excitation of multiple neurons and of glial cells. Methods/Principal Findings The system combines a liquid crystal device for holographic patterned photostimulation, high-resolution optical imaging, the HiLo microscopy, to define the stimulated regions and a conventional Ca2+ imaging system to detect neural activity. By means of electrophysiological recordings and calcium imaging in acute hippocampal slices, we show that the use of excitation patterns precisely tailored to the shape of multiple neuronal somata represents a very efficient way for the simultaneous excitation of a group of neurons. In addition, we demonstrate that fast shaped illumination patterns also induce reliable responses in single glial cells. Conclusions/Significance We show that the main advantage of holographic illumination is that it allows for an efficient excitation of multiple cells with a spatiotemporal resolution unachievable with other existing approaches. Although this paper focuses on the photoactivation of caged molecules, our approach will surely prove very efficient for other probes, such as light-gated channels, genetically encoded photoactivatable proteins, photoactivatable fluorescent proteins, and voltage-sensitive dyes. PMID:20195547

Novel advancements in colposcopy: historical perspectives and a systematic review of future developments.

PubMed

Adelman, Marisa Rachel

2014-07-01

To describe novel innovations and techniques for the detection of high-grade dysplasia. Studies were identified through the PubMed database, spanning the last 10 years. The key words (["computerized colposcopy" or "digital colposcopy" or "spectroscopy" or "multispectral digital colposcopy" or "dynamic spectral imaging", or "electrical impedance spectroscopy" or "confocal endomicroscopy" or "confocal microscopy"or "optical coherence tomography"] and ["cervical dysplasia" or cervical precancer" or "cervix" or "cervical"]) were used. The inclusion criteria were published articles of original research referring to noncolposcopic evaluation of the cervix for the detection of cervical dysplasia. Only English-language articles from the past 10 years were included, in which the technologies were used in vivo, and sensitivities and specificities could be calculated. The single author reviewed the articles for inclusion. Primary search of the database yielded 59 articles, and secondary cross-reference yielded 12 articles. Thirty-two articles met the inclusion criteria. An instrument that globally assesses the cervix, such as computer-assisted colposcopy, optical spectroscopy, and dynamic spectral imaging, would provided the most comprehensive estimate of disease and is therefore best suited when treatment is preferred. Electrical impedance spectroscopy, confocal microscopy, and optical coherence tomography provide information at the cellular level to estimate histology and are therefore best suited when deferment of treatment is preferred. If a device is to eventually replace the colposcope, it will likely combine technologies to best meet the needs of the target population, and as such, no single instrument may prove to be universally appropriate. Analyses of false-positive rates, additional colposcopies and biopsies, cost, and absolute life-savings will be important when considering these technologies and are limited thus far.

CXRO - Mi-Young Im, Staff Scientist

Science.gov Websites

X-Ray Database Zone Plate Education Nanomagnetism X-Ray Microscopy LDJIM EUV Lithography EUV Mask Publications Contact The Center for X-Ray Optics is a multi-disciplined research group within Lawrence Berkeley -Ray Optics X-Ray Database Nanomagnetism X-Ray Microscopy EUV Lithography EUV Mask Imaging

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.

Spatially and temporally resolved exciton dynamics and transport in single nanostructures and assemblies

NASA Astrophysics Data System (ADS)

Huang, Libai

2015-03-01

The frontier in solar energy conversion now lies in learning how to integrate functional entities across multiple length scales to create optimal devices. To address this new frontier, I will discuss our recent efforts on elucidating multi-scale energy transfer, migration, and dissipation processes with simultaneous femtosecond temporal resolution and nanometer spatial resolution. We have developed ultrafast microscopy that combines ultrafast spectroscopy with optical microscopy to map exciton dynamics and transport with simultaneous ultrafast time resolution and diffraction-limited spatial resolution. We have employed pump-probe transient absorption microscopy to elucidate morphology and structure dependent exciton dynamics and transport in single nanostructures and molecular assemblies. More specifically, (1) We have applied transient absorption microscopy (TAM) to probe environmental and structure dependent exciton relaxation pathways in sing-walled carbon nanotubes (SWNTs) by mapping dynamics in individual pristine SWNTs with known structures. (2) We have systematically measured and modeled the optical properties of the Frenkel excitons in self-assembled porphyrin tubular aggregates that represent an analog to natural photosynthetic antennae. Using a combination of ultrafast optical microscopy and stochastic exciton modeling, we address exciton transport and relaxation pathways, especially those related to disorder.

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

Scene-based Shack-Hartmann wavefront sensor for light-sheet microscopy

NASA Astrophysics Data System (ADS)

Lawrence, Keelan; Liu, Yang; Dale, Savannah; Ball, Rebecca; VanLeuven, Ariel J.; Sornborger, Andrew; Lauderdale, James D.; Kner, Peter

2018-02-01

Light-sheet microscopy is an ideal imaging modality for long-term live imaging in model organisms. However, significant optical aberrations can be present when imaging into an organism that is hundreds of microns or greater in size. To measure and correct optical aberrations, an adaptive optics system must be incorporated into the microscope. Many biological samples lack point sources that can be used as guide stars with conventional Shack-Hartmann wavefront sensors. We have developed a scene-based Shack-Hartmann wavefront sensor for measuring the optical aberrations in a light-sheet microscopy system that does not require a point-source and can measure the aberrations for different parts of the image. The sensor has 280 lenslets inside the pupil, creates an image from each lenslet with a 500 micron field of view and a resolution of 8 microns, and has a resolution for the wavefront gradient of 75 milliradians per lenslet. We demonstrate the system on both fluorescent bead samples and zebrafish embryos.

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

Effect of composition and temperature on the second harmonic generation in silver phosphate glasses

NASA Astrophysics Data System (ADS)

Konidakis, I.; Psilodimitrakopoulos, S.; Kosma, K.; Lemonis, A.; Stratakis, E.

2018-01-01

We herein employ nonlinear laser imaging microscopy to explicitly study the dynamics of second harmonic generation (SHG) in silver iodide phosphate glasses. While glasses of this family have gained extensive scientific attention over the years due to their superior conducting properties, considerably less attention has been paid to their unique nonlinear optical characteristics. In the present study, firstly, it is demonstrated that SHG signal intensity is enhanced upon increasing silver content due to the random formation of silver microstructures within the glass network. Secondly, the SHG temperature dynamics were explored near the glass transition temperature (Tg) regime, where significant glass relaxation phenomena occur. It is found that heating towards the Tg improves the SHG efficiency, whereas above Tg, the capacity of glasses to generate second harmonic radiation is drastically suppressed. The novel findings of this work are considered important in terms of the potential employment of these glasses for the realization of advanced photonic applications like optical-switches and wavelength conversion devices.

Nanoscale Characterization of Carrier Dynamic and Surface Passivation in InGaN/GaN Multiple Quantum Wells on GaN Nanorods.

PubMed

Chen, Weijian; Wen, Xiaoming; Latzel, Michael; Heilmann, Martin; Yang, Jianfeng; Dai, Xi; Huang, Shujuan; Shrestha, Santosh; Patterson, Robert; Christiansen, Silke; Conibeer, Gavin

2016-11-23

Using advanced two-photon excitation confocal microscopy, associated with time-resolved spectroscopy, we characterize InGaN/GaN multiple quantum wells on nanorod heterostructures and demonstrate the passivation effect of a KOH treatment. High-quality InGaN/GaN nanorods were fabricated using nanosphere lithography as a candidate material for light-emitting diode devices. The depth- and time-resolved characterization at the nanoscale provides detailed carrier dynamic analysis helpful for understanding the optical properties. The nanoscale spatially resolved images of InGaN quantum well and defects were acquired simultaneously. We demonstrate that nanorod etching improves light extraction efficiency, and a proper KOH treatment has been found to reduce the surface defects efficiently and enhance the luminescence. The optical characterization techniques provide depth-resolved and time-resolved carrier dynamics with nanoscale spatially resolved mapping, which is crucial for a comprehensive and thorough understanding of nanostructured materials and provides novel insight into the improvement of materials fabrication and applications.

Detection of quantum well induced single degenerate-transition-dipoles in ZnO nanorods.

PubMed

Ghosh, Siddharth; Ghosh, Moumita; Seibt, Michael; Rao, G Mohan

2016-02-07

Quantifying and characterising atomic defects in nanocrystals is difficult and low-throughput using the existing methods such as high resolution transmission electron microscopy (HRTEM). In this article, using a defocused wide-field optical imaging technique, we demonstrate that a single ultrahigh-piezoelectric ZnO nanorod contains a single defect site. We model the observed dipole-emission patterns from optical imaging with a multi-dimensional dipole and find that the experimentally observed dipole pattern and model-calculated patterns are in excellent agreement. This agreement suggests the presence of vertically oriented degenerate-transition-dipoles in vertically aligned ZnO nanorods. The HRTEM of the ZnO nanorod shows the presence of a stacking fault, which generates a localised quantum well induced degenerate-transition-dipole. Finally, we elucidate that defocused wide-field imaging can be widely used to characterise defects in nanomaterials to answer many difficult questions concerning the performance of low-dimensional devices, such as in energy harvesting, advanced metal-oxide-semiconductor storage, and nanoelectromechanical and nanophotonic devices.

Superior electro-optical properties of electrically controlled birefringence mode using solution-derived La{sub 2}O{sub 3} films

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeong, Hae-Chang; Park, Hong-Gyu; Lee, Ju Hwan

2015-11-15

The authors demonstrate a high performance electrically controlled birefringence (ECB) mode with solution-derived La{sub 2}O{sub 3} films at various molar concentrations. Uniform and homogeneous liquid crystal (LC) alignment was spontaneously achieved on the La{sub 2}O{sub 3} films for lanthanum concentrations at ratios greater than and equal to 0.2. A preferred orientation of LC molecules appeared along the filling direction, and the LC alignment was maintained via van der Waals force by nanocrystals of the La{sub 2}O{sub 3} films. The LC alignment mechanism was confirmed by x-ray photoelectron spectroscopy and high-resolution transmission electron microscopy analysis. Superior electro-optical characteristics of the ECBmore » cells constructed with solution-derived La{sub 2}O{sub 3} films were observed, which suggests that the proposed solution-derived La{sub 2}O{sub 3} films have strong potential for use in the production of advanced LC displays.« less

Optical coherence tomography for embryonic imaging: a review

PubMed Central

Raghunathan, Raksha; Singh, Manmohan; Dickinson, Mary E.; Larin, Kirill V.

2016-01-01

Abstract. Embryogenesis is a highly complex and dynamic process, and its visualization is crucial for understanding basic physiological processes during development and for identifying and assessing possible defects, malformations, and diseases. While traditional imaging modalities, such as ultrasound biomicroscopy, micro-magnetic resonance imaging, and micro-computed tomography, have long been adapted for embryonic imaging, these techniques generally have limitations in their speed, spatial resolution, and contrast to capture processes such as cardiodynamics during embryogenesis. Optical coherence tomography (OCT) is a noninvasive imaging modality with micrometer-scale spatial resolution and imaging depth up to a few millimeters in tissue. OCT has bridged the gap between ultrahigh resolution imaging techniques with limited imaging depth like confocal microscopy and modalities, such as ultrasound sonography, which have deeper penetration but poorer spatial resolution. Moreover, the noninvasive nature of OCT has enabled live imaging of embryos without any external contrast agents. We review how OCT has been utilized to study developing embryos and also discuss advances in techniques used in conjunction with OCT to understand embryonic development. PMID:27228503

Probing the ultimate plasmon confinement limits with a van der Waals heterostructure.

PubMed

Alcaraz Iranzo, David; Nanot, Sébastien; Dias, Eduardo J C; Epstein, Itai; Peng, Cheng; Efetov, Dmitri K; Lundeberg, Mark B; Parret, Romain; Osmond, Johann; Hong, Jin-Yong; Kong, Jing; Englund, Dirk R; Peres, Nuno M R; Koppens, Frank H L

2018-04-20

The ability to confine light into tiny spatial dimensions is important for applications such as microscopy, sensing, and nanoscale lasers. Although plasmons offer an appealing avenue to confine light, Landau damping in metals imposes a trade-off between optical field confinement and losses. We show that a graphene-insulator-metal heterostructure can overcome that trade-off, and demonstrate plasmon confinement down to the ultimate limit of the length scale of one atom. This is achieved through far-field excitation of plasmon modes squeezed into an atomically thin hexagonal boron nitride dielectric spacer between graphene and metal rods. A theoretical model that takes into account the nonlocal optical response of both graphene and metal is used to describe the results. These ultraconfined plasmonic modes, addressed with far-field light excitation, enable a route to new regimes of ultrastrong light-matter interactions. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Pump-probe optical microscopy for imaging nonfluorescent chromophores.

PubMed

Wei, Lu; Min, Wei

2012-06-01

Many chromophores absorb light intensely but have undetectable fluorescence. Hence microscopy techniques other than fluorescence are highly desirable for imaging these chromophores inside live cells, tissues, and organisms. The recently developed pump-probe optical microscopy techniques provide fluorescence-free contrast mechanisms by employing several fundamental light-molecule interactions including excited state absorption, stimulated emission, ground state depletion, and the photothermal effect. By using the pump pulse to excite molecules and the subsequent probe pulse to interrogate the created transient states on a laser scanning microscope, pump-probe microscopy offers imaging capability with high sensitivity and specificity toward nonfluorescent chromophores. Single-molecule sensitivity has even been demonstrated. Here we review and summarize the underlying principles of this emerging class of molecular imaging techniques.

Optical imaging beyond the diffraction limit by SNEM: effects of AFM tip modifications with thiol monolayers on imaging quality.

PubMed

Cumurcu, Aysegul; Diaz, Jordi; Lindsay, Ian D; de Beer, Sissi; Duvigneau, Joost; Schön, Peter; Julius Vancso, G

2015-03-01

Tip-enhanced nanoscale optical imaging techniques such as apertureless scanning near-field optical microscopy (a-SNOM) and scanning near-field ellipsometric microscopy (SNEM) applications can suffer from a steady degradation in performance due to adhesion of atmospheric contaminants to the metal coated tip. Here, we demonstrate that a self-assembled monolayer (SAM) of ethanethiol (EtSH) is an effective means of protecting gold-coated atomic force microscopy (AFM) probe tips from accumulation of surface contaminants during prolonged exposure to ambient air. The period over which they yield consistent and reproducible results for scanning near-field ellipsometric microscopy (SNEM) imaging is thus extended. SNEM optical images of a microphase separated polystyrene-block-poly (methylmethacrylate) (PS-b-PMMA) diblock copolymer film, which were captured with bare and SAM-protected gold-coated AFM probes, both immediately after coating and following five days of storage in ambient air, were compared. During this period the intensity of the optical signals from the untreated gold tip fell by 66%, while those from the SAM protected tip fell by 14%. Additionally, gold coated AFM probe tips were modified with various lengths of alkanethiols to measure the change in intensity variation in the optical images with SAM layer thickness. The experimental results were compared to point dipole model calculations. While a SAM of 1-dodecanethiol (DoSH) was found to strongly suppress field enhancement we find that it can be locally removed from the tip apex by deforming the molecules under load, restoring SNEM image contrast. Copyright © 2014 Elsevier B.V. All rights reserved.

Computational adaptive optics for broadband optical interferometric tomography of biological tissue.

PubMed

Adie, Steven G; Graf, Benedikt W; Ahmad, Adeel; Carney, P Scott; Boppart, Stephen A

2012-05-08

Aberrations in optical microscopy reduce image resolution and contrast, and can limit imaging depth when focusing into biological samples. Static correction of aberrations may be achieved through appropriate lens design, but this approach does not offer the flexibility of simultaneously correcting aberrations for all imaging depths, nor the adaptability to correct for sample-specific aberrations for high-quality tomographic optical imaging. Incorporation of adaptive optics (AO) methods have demonstrated considerable improvement in optical image contrast and resolution in noninterferometric microscopy techniques, as well as in optical coherence tomography. Here we present a method to correct aberrations in a tomogram rather than the beam of a broadband optical interferometry system. Based on Fourier optics principles, we correct aberrations of a virtual pupil using Zernike polynomials. When used in conjunction with the computed imaging method interferometric synthetic aperture microscopy, this computational AO enables object reconstruction (within the single scattering limit) with ideal focal-plane resolution at all depths. Tomographic reconstructions of tissue phantoms containing subresolution titanium-dioxide particles and of ex vivo rat lung tissue demonstrate aberration correction in datasets acquired with a highly astigmatic illumination beam. These results also demonstrate that imaging with an aberrated astigmatic beam provides the advantage of a more uniform depth-dependent signal compared to imaging with a standard gaussian beam. With further work, computational AO could enable the replacement of complicated and expensive optical hardware components with algorithms implemented on a standard desktop computer, making high-resolution 3D interferometric tomography accessible to a wider group of users and nonspecialists.

Fabrication and characterization of a nanometer-sized optical fiber electrode based on selective chemical etching for scanning electrochemical/optical microscopy.

PubMed

Maruyama, Kenichi; Ohkawa, Hiroyuki; Ogawa, Sho; Ueda, Akio; Niwa, Osamu; Suzuki, Koji

2006-03-15

We have already reported a method for fabricating ultramicroelectrodes (Suzuki, K. JP Patent, 2004-45394, 2004). This method is based on the selective chemical etching of optical fibers. In this work, we undertake a detailed investigation involving a combination of etched optical fibers with various types of tapered tip (protruding-shape, double- (or pencil-) shape and triple-tapered electrode) and insulation with electrophoretic paint. Our goal is to establish a method for fabricating nanometer-sized optical fiber electrodes with high reproducibility. As a result, we realized pencil-shaped and triple-tapered electrodes that had radii in the nanometer range with high reproducibility. These nanometer-sized electrodes showed well-defined sigmoidal curves and stable diffusion-limited responses with cyclic voltammetry. The pencil-shaped optical fiber, which has a conical tip with a cone angle of 20 degrees , was effective for controlling the electrode radius. The pencil-shaped electrodes had higher reproducibility and smaller electrode radii (r(app) < 1.0 nm) than those of other etched optical fiber electrodes. By using a pencil-shaped electrode with a 105-nm radius as a probe, we obtained simultaneous electrochemical and optical images of an implantable interdigitated array electrode. We achieved nanometer-scale resolution with a combination of scanning electrochemical microscopy SECM and optical microscopy. The resolution of the electrochemical and optical images indicated sizes of 300 and 930 nm, respectively. The neurites of living PC12 cells were also successfully imaged on a 1.6-microm scale by using the negative feedback mode of an SECM.

Sensitivity of photoacoustic microscopy

PubMed Central

Yao, Junjie; Wang, Lihong V.

2014-01-01

Building on its high spatial resolution, deep penetration depth and excellent image contrast, 3D photoacoustic microscopy (PAM) has grown tremendously since its first publication in 2005. Integrating optical excitation and acoustic detection, PAM has broken through both the optical diffusion and optical diffraction limits. PAM has 100% relative sensitivity to optical absorption (i.e., a given percentage change in the optical absorption coefficient yields the same percentage change in the photoacoustic amplitude), and its ultimate detection sensitivity is limited only by thermal noise. Focusing on the engineering aspects of PAM, this Review discusses the detection sensitivity of PAM, compares the detection efficiency of different PAM designs, and summarizes the imaging performance of various endogenous and exogenous contrast agents. It then describes representative PAM applications with high detection sensitivity, and outlines paths to further improvement. PMID:25302158

Back to the Future - Part 1. The medico-legal autopsy from ancient civilization to the post-genomic era.

PubMed

Cecchetto, Giovanni; Bajanowski, Thomas; Cecchi, Rossana; Favretto, Donata; Grabherr, Silke; Ishikawa, Takaki; Kondo, Toshikazu; Montisci, Massimo; Pfeiffer, Heidi; Bonati, Maurizio Rippa; Shokry, Dina; Vennemann, Marielle; Ferrara, Santo Davide

2017-07-01

Part 1 of the review "Back to the Future" examines the historical evolution of the medico-legal autopsy and microscopy techniques, from Ancient Civilization to the Post-Genomic Era. In the section focusing on "The Past", the study of historical sources concerning the origins and development of the medico-legal autopsy, from the Bronze Age until the Middle Ages, shows how, as early as 2000 BC, the performance of autopsies for medico-legal purposes was a known and widespread practice in some ancient civilizations in Egypt, the Far East and later in Europe. In the section focusing on "The Present", the improvement of autopsy techniques by Friedrich Albert Zenker and Rudolf Virchow and the contemporary development of optical microscopy techniques for forensic purposes during the 19th and 20th centuries are reported, emphasizing, the regulation of medico-legal autopsies in diverse nations around the world and the publication of international guidelines or best practices elaborated by International Scientific Societies. Finally, in "The Future" section, innovative robotized and advanced microscopy systems and techniques, including their possible use in the bio-medicolegal field, are reported, which should lead to the improvement and standardization of the autopsy methodology, thereby achieving a more precise identification of natural and traumatic pathologies.

Breaking resolution limits in ultrafast electron diffraction and microscopy.

PubMed

Baum, Peter; Zewail, Ahmed H

2006-10-31

Ultrafast electron microscopy and diffraction are powerful techniques for the study of the time-resolved structures of molecules, materials, and biological systems. Central to these approaches is the use of ultrafast coherent electron packets. The electron pulses typically have an energy of 30 keV for diffraction and 100-200 keV for microscopy, corresponding to speeds of 33-70% of the speed of light. Although the spatial resolution can reach the atomic scale, the temporal resolution is limited by the pulse width and by the difference in group velocities of electrons and the light used to initiate the dynamical change. In this contribution, we introduce the concept of tilted optical pulses into diffraction and imaging techniques and demonstrate the methodology experimentally. These advances allow us to reach limits of time resolution down to regimes of a few femtoseconds and, possibly, attoseconds. With tilted pulses, every part of the sample is excited at precisely the same time as when the electrons arrive at the specimen. Here, this approach is demonstrated for the most unfavorable case of ultrafast crystallography. We also present a method for measuring the duration of electron packets by autocorrelating electron pulses in free space and without streaking, and we discuss the potential of tilting the electron pulses themselves for applications in domains involving nuclear and electron motions.

Determination of the resolution of the x-ray microscope XM-1 at beamline 6.1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heck, J.M.; Meyer-Ilse, W.; Attwood, D.T.

1997-04-01

Resolution determination in x-ray microscopy is a complex issue which depends on many factors. Many different criteria and experimental setups are used to characterize resolution. Some of the important factors affecting resolution include the partial coherence and spectrum of the illumination. The purpose of this research has been to measure the resolution of XM-1 at beamline 6.1 taking into account these factors, and to compare the measurements to theoretical calculations. The x-ray microscope XM-1, built by the Center for X-ray Optics (CXRO), has been operational since 1994 at the Advanced Light Source at E.O. Lawrence Berkeley National Laboratory. It ismore » of the conventional (i.e. full-field) type, utilizing zone plate optics. ALS bending magnet radiation is focused by a condenser zone plate onto a monochromator pinhole immediately in front of the sample. X-rays transmitted through the sample are focused by a micro-zone plate onto a CCD camera. The pinhole and the condenser with a central stop constitute a linear monochromator. The spectral distribution of the light illuminating the sample has been calculated assuming geometrical optics.« less

Nanofinishing of BK7 glass using a magnetorheological solid rotating core tool.

PubMed

Kumar, Sumit; Singh, Anant Kumar

2018-02-01

Surface finishing is a promising method to improve the optical characteristics of crown glass. BK7 finds its applications in transmissive optics, i.e., lenses of binoculars, lenses of microscopes, lenses of telescopes, and light-emitting diodes. The magnetorheological (MR) nanofinishing of optical glasses using a solid rotating core tool is found more advantageous than the other advanced finishing processes in aspects such as precision and accuracy. In the present research, the MR nanofinishing with a solid rotating core tool is carried out on the BK7 glass of size 10×10×3  mm. Response surface methodology is conducted in order to find the optimum process parameters. The effects of process parameters on the percentage change in surface roughness are analyzed. The best surface roughness R a and R q values are achieved at 22 nm and 32 nm from the initial of 41 nm and 57 nm in 30 min of the finishing time cycle. To study the surface morphology of nanofinished BK7 glass, scanning electron microscopy is performed with sputter coating of gold on a glass specimen.

Neurite density from magnetic resonance diffusion measurements at ultrahigh field: Comparison with light microscopy and electron microscopy

PubMed Central

Jespersen, Sune N.; Bjarkam, Carsten R.; Nyengaard, Jens R.; Chakravarty, M. Mallar; Hansen, Brian; Vosegaard, Thomas; Østergaard, Leif; Yablonskiy, Dmitriy; Nielsen, Niels Chr.; Vestergaard-Poulsen, Peter

2010-01-01

Due to its unique sensitivity to tissue microstructure, diffusion-weighted magnetic resonance imaging (MRI) has found many applications in clinical and fundamental science. With few exceptions, a more precise correspondence between physiological or biophysical properties and the obtained diffusion parameters remain uncertain due to lack of specificity. In this work, we address this problem by comparing diffusion parameters of a recently introduced model for water diffusion in brain matter to light microscopy and quantitative electron microscopy. Specifically, we compare diffusion model predictions of neurite density in rats to optical myelin staining intensity and stereological estimation of neurite volume fraction using electron microscopy. We find that the diffusion model describes data better and that its parameters show stronger correlation with optical and electron microscopy, and thus reflect myelinated neurite density better than the more frequently used diffusion tensor imaging (DTI) and cumulant expansion methods. Furthermore, the estimated neurite orientations capture dendritic architecture more faithfully than DTI diffusion ellipsoids. PMID:19732836

In vivo correlation mapping microscopy

NASA Astrophysics Data System (ADS)

McGrath, James; Alexandrov, Sergey; Owens, Peter; Subhash, Hrebesh; Leahy, Martin

2016-04-01

To facilitate regular assessment of the microcirculation in vivo, noninvasive imaging techniques such as nailfold capillaroscopy are required in clinics. Recently, a correlation mapping technique has been applied to optical coherence tomography (OCT), which extends the capabilities of OCT to microcirculation morphology imaging. This technique, known as correlation mapping optical coherence tomography, has been shown to extract parameters, such as capillary density and vessel diameter, and key clinical markers associated with early changes in microvascular diseases. However, OCT has limited spatial resolution in both the transverse and depth directions. Here, we extend this correlation mapping technique to other microscopy modalities, including confocal microscopy, and take advantage of the higher spatial resolution offered by these modalities. The technique is achieved as a processing step on microscopy images and does not require any modification to the microscope hardware. Results are presented which show that this correlation mapping microscopy technique can extend the capabilities of conventional microscopy to enable mapping of vascular networks in vivo with high spatial resolution in both the transverse and depth directions.

Characterization of konjac glucomannan-ethyl cellulose film formation via microscopy.

PubMed

Xiao, Man; Wan, Li; Corke, Harold; Yan, Wenli; Ni, Xuewen; Fang, Yapeng; Jiang, Fatang

2016-04-01

Konjac glucomannan-ethyl cellulose (KGM-EC, 7:3, w/w) blended film shows good mechanical and moisture resistance properties. To better understand the basis for the KGM-EC film formation, optical microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) were used to observe the formation of the film from emulsion. Optical microscopy images showed that EC oil droplets were homogeneously dispersed in KGM water phase without obviously coalescence throughout the entire drying process. SEM images showed the surface and cross-sectional structures of samples maintained continuous and homogeneous appearance from the emulsion to dried film. AFM images indicated that KGM molecules entangled EC molecules in the emulsion. Interactions between KGM and EC improved the stability of KGM-EC emulsion, and contributed to uniformed structures of film formation. Based on these output information, a schematic model was built to elucidate KGM-EC film-forming process. Copyright © 2015 Elsevier B.V. All rights reserved.

A rapid approach to high-resolution fluorescence imaging in semi-thick brain slices.

PubMed

Selever, Jennifer; Kong, Jian-Qiang; Arenkiel, Benjamin R

2011-07-26

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps(1-3). With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks(4). Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will(5-6). Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates(7-8), alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters(9). Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision. Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods(10), or 2-photon laser scanning microscopy(11). Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively. Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1). For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Wide field fluorescence epi-microscopy behind a scattering medium enabled by speckle correlations

NASA Astrophysics Data System (ADS)

Hofer, Matthias; Soeller, Christian; Brasselet, Sophie; Bertolotti, Jacopo

2018-04-01

Fluorescence microscopy is widely used in biological imaging, however scattering from tissues strongly limits its applicability to a shallow depth. In this work we adapt a methodology inspired from stellar speckle interferometry, and exploit the optical memory effect to enable fluorescence microscopy through a turbid layer. We demonstrate efficient reconstruction of micrometer-size fluorescent objects behind a scattering medium in epi-microscopy, and study the specificities of this imaging modality (magnification, field of view, resolution) as compared to traditional microscopy. Using a modified phase retrieval algorithm to reconstruct fluorescent objects from speckle images, we demonstrate robust reconstructions even in relatively low signal to noise conditions. This modality is particularly appropriate for imaging in biological media, which are known to exhibit relatively large optical memory ranges compatible with tens of micrometers size field of views, and large spectral bandwidths compatible with emission fluorescence spectra of tens of nanometers widths.

Optical properties of mouse brain tissue after optical clearing with FocusClear™

NASA Astrophysics Data System (ADS)

Moy, Austin J.; Capulong, Bernard V.; Saager, Rolf B.; Wiersma, Matthew P.; Lo, Patrick C.; Durkin, Anthony J.; Choi, Bernard

2015-09-01

Fluorescence microscopy is commonly used to investigate disease progression in biological tissues. Biological tissues, however, are strongly scattering in the visible wavelengths, limiting the application of fluorescence microscopy to superficial (<200 μm) regions. Optical clearing, which involves incubation of the tissue in a chemical bath, reduces the optical scattering in tissue, resulting in increased tissue transparency and optical imaging depth. The goal of this study was to determine the time- and wavelength-resolved dynamics of the optical scattering properties of rodent brain after optical clearing with FocusClear™. Light transmittance and reflectance of 1-mm mouse brain sections were measured using an integrating sphere before and after optical clearing and the inverse adding doubling algorithm used to determine tissue optical scattering. The degree of optical clearing was quantified by calculating the optical clearing potential (OCP), and the effects of differing OCP were demonstrated using the optical histology method, which combines tissue optical clearing with optical imaging to visualize the microvasculature. We observed increased tissue transparency with longer optical clearing time and an analogous increase in OCP. Furthermore, OCP did not vary substantially between 400 and 1000 nm for increasing optical clearing durations, suggesting that optical histology can improve ex vivo visualization of several fluorescent probes.

Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection

PubMed Central

Zhi, Yanan; Wang, Benquan; Yao, Xincheng

2016-01-01

Light microscopy plays a key role in biological studies and medical diagnosis. The spatial resolution of conventional optical microscopes is limited to approximately half the wavelength of the illumination light as a result of the diffraction limit. Several approaches—including confocal microscopy, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy, and structured illumination microscopy—have been established to achieve super-resolution imaging. However, none of these methods is suitable for the super-resolution ophthalmoscopy of retinal structures because of laser safety issues and inevitable eye movements. We recently experimentally validated virtually structured detection (VSD) as an alternative strategy to extend the diffraction limit. Without the complexity of structured illumination, VSD provides an easy, low-cost, and phase artifact–free strategy to achieve super-resolution in scanning laser microscopy. In this article we summarize the basic principles of the VSD method, review our demonstrated single-point and line-scan super-resolution systems, and discuss both technical challenges and the potential of VSD-based instrumentation for super-resolution ophthalmoscopy of the retina. PMID:27480461

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.

Cytology 3D structure formation based on optical microscopy images

NASA Astrophysics Data System (ADS)

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

2017-01-01

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

Silicon technology-based micro-systems for atomic force microscopy/photon scanning tunnelling microscopy.

PubMed

Gall-Borrut, P; Belier, B; Falgayrettes, P; Castagne, M; Bergaud, C; Temple-Boyer, P

2001-04-01

We developed silicon nitride cantilevers integrating a probe tip and a wave guide that is prolonged on the silicon holder with one or two guides. A micro-system is bonded to a photodetector. The resulting hybrid system enables us to obtain simultaneously topographic and optical near-field images. Examples of images obtained on a longitudinal cross-section of an optical fibre are shown.

Microcontact Printing via a Polymer-Induced Liquid-Precursor (PILP) Process

DTIC Science & Technology

2002-04-01

applications that require high performance mechanical, electrical and/or optical properties resulting from controlled nano- and microstructural design...salts. The cover-slips were examined by optical microscopy, and then gold coated for scanning electron microscopy on a SEM JEOL JSM 6400 instrument [5...applications in the realm of biomimicry . Controlled growth of crystals with specific orientation can be achieved via the functional groups on the substrate

Orbital angular momentum light in microscopy

PubMed Central

2017-01-01

Light with a helical phase has had an impact on optical imaging, pushing the limits of resolution or sensitivity. Here, special emphasis will be given to classical light microscopy of phase samples and to Fourier filtering techniques with a helical phase profile, such as the spiral phase contrast technique in its many variants and areas of application. This article is part of the themed issue ‘Optical orbital angular momentum’. PMID:28069768

In-Fiber Magneto-Optic Devices Based on Ultrahigh Verdet Constant Organic Materials and Holey Fibers

DTIC Science & Technology

2009-02-02

protocols and a noise equivalent magnetic field sensitivity of ~ 100 pT/ VHz has been demonstrated. • Magneto-optic properties of magnetite - PMMA composite...nanoparticle - PMMA nanocomposite. We have used both transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) to...we expect to enhance it in our devices by their proper symmetrization as described above. Passive Poking core ^^ direction Magnetic AA

Optical Biomarkers of Serous and Mucinous Human Ovarian Tumor Assessed with Nonlinear Optics Microscopies

PubMed Central

Adur, Javier; Pelegati, Vitor B.; de Thomaz, Andre A.; Baratti, Mariana O.; Almeida, Diogo B.; Andrade, L. A. L. A.; Bottcher-Luiz, Fátima; Carvalho, Hernandes F.; Cesar, Carlos L.

2012-01-01

Background Nonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression. Methodology/Principal Findings We obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells. Conclusions/Significance NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions. PMID:23056557

Spatiotemporal polarization modulation microscopy with a microretarder array

NASA Astrophysics Data System (ADS)

Ding, Changqin; Ulcickas, James R. W.; Simpson, Garth J.

2018-02-01

A patterned microretarder array positioned in the rear conjugate plane of a microscope enables rapid polarizationdependent nonlinear optical microscopy. The pattern introduced to the array results in periodic modulation of the polarization-state of the incident light as a function of position within the field of view with no moving parts or active control. Introduction of a single stationary optical element and a fixed polarizer into the beam of a nonlinear optical microscope enabled nonlinear optical tensor recovery, which informs on local structure and orientation. Excellent agreement was observed between the measured and predicted second harmonic generation (SHG) of z-cut quartz, selected as a test system with well-established nonlinear optical properties. Subsequent studies of spatially varying samples further support the general applicability of this relatively simple strategy for detailed polarization analysis in both conventional and nonlinear optical imaging of structurally diverse samples.

Center for Adaptive Optics | Events

Science.gov Websites

Center for Adaptive Optics A University of California Science and Technology Center home 2015 AO Adaptive Optics and Wavefront Control in Microscopy and Ophthalmology Paris, France October 25-25 CfAO Adaptive Optics Institute for Scientist and Engineer Educators Members Calendar of Events Publications

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

PubMed

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

2016-03-10

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

Optically coupled methods for microwave impedance microscopy

NASA Astrophysics Data System (ADS)

Johnston, Scott R.; Ma, Eric Yue; Shen, Zhi-Xun

2018-04-01

Scanning Microwave Impedance Microscopy (MIM) measurement of photoconductivity with 50 nm resolution is demonstrated using a modulated optical source. The use of a modulated source allows for the measurement of photoconductivity in a single scan without a reference region on the sample, as well as removing most topographical artifacts and enhancing signal to noise as compared with unmodulated measurement. A broadband light source with a tunable monochrometer is then used to measure energy resolved photoconductivity with the same methodology. Finally, a pulsed optical source is used to measure local photo-carrier lifetimes via MIM, using the same 50 nm resolution tip.

Development of Nomarski microscopy for quantitative determination of surface topography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hartman, J. S.; Gordon, R. L.; Lessor, D. L.

1979-01-01

The use of Nomarski differential interference contrast (DIC) microscopy has been extended to provide nondestructive, quantitative analysis of a sample's surface topography. Theoretical modeling has determined the dependence of the image intensity on the microscope's optical components, the sample's optical properties, and the sample's surface orientation relative to the microscope. Results include expressions to allow the inversion of image intensity data to determine sample surface slopes. A commercial Nomarski system has been modified and characterized to allow the evaluation of the optical model. Data have been recorded with smooth, planar samples that verify the theoretical predictions.

Axial Colocalization of Single Molecules with Nanometer Accuracy Using Metal-Induced Energy Transfer.

PubMed

Isbaner, Sebastian; Karedla, Narain; Kaminska, Izabela; Ruhlandt, Daja; Raab, Mario; Bohlen, Johann; Chizhik, Alexey; Gregor, Ingo; Tinnefeld, Philip; Enderlein, Jörg; Tsukanov, Roman

2018-04-11

Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups.

In vivo confocal microscopy of the cornea: New developments in image acquisition, reconstruction and analysis using the HRT-Rostock Corneal Module

PubMed Central

Petroll, W. Matthew; Robertson, Danielle M.

2015-01-01

The optical sectioning ability of confocal microscopy allows high magnification images to be obtained from different depths within a thick tissue specimen, and is thus ideally suited to the study of intact tissue in living subjects. In vivo confocal microscopy has been used in a variety of corneal research and clinical applications since its development over 25 years ago. In this article we review the latest developments in quantitative corneal imaging with the Heidelberg Retinal Tomograph with Rostock Corneal Module (HRT-RCM). We provide an overview of the unique strengths and weaknesses of the HRT-RCM. We discuss techniques for performing 3-D imaging with the HRT-RCM, including hardware and software modifications that allow full thickness confocal microscopy through focusing (CMTF) of the cornea, which can provide quantitative measurements of corneal sublayer thicknesses, stromal cell and extracellular matrix backscatter, and depth dependent changes in corneal keratocyte density. We also review current approaches for quantitative imaging of the subbasal nerve plexus, which require a combination of advanced image acquisition and analysis procedures, including wide field mapping and 3-D reconstruction of nerve structures. The development of new hardware, software, and acquisition techniques continues to expand the number of applications of the HRT-RCM for quantitative in vivo corneal imaging at the cellular level. Knowledge of these rapidly evolving strategies should benefit corneal clinicians and basic scientists alike. PMID:25998608

Techniques for 3D tracking of single molecules with nanometer accuracy in living cells

NASA Astrophysics Data System (ADS)

Gardini, Lucia; Capitanio, Marco; Pavone, Francesco S.

2013-06-01

We describe a microscopy technique that, combining wide-field single molecule microscopy, bifocal imaging and Highly Inclined and Laminated Optical sheet (HILO) microscopy, allows a 3D tracking with nanometer accuracy of single fluorescent molecules in vitro and in living cells.

Simple fiber-optic confocal microscopy with nanoscale depth resolution beyond the diffraction barrier.

PubMed

Ilev, Ilko; Waynant, Ronald; Gannot, Israel; Gandjbakhche, Amir

2007-09-01

A novel fiber-optic confocal approach for ultrahigh depth-resolution (

Functional Micrococcus lysodeikticus layers deposited by laser technique for the optical sensing of lysozyme.

PubMed

Dinca, Valentina; Zaharie-Butucel, Diana; Stanica, Luciana; Brajnicov, Simona; Marascu, Valentina; Bonciu, Anca; Cristocea, Andra; Gaman, Laura; Gheorghiu, Mihaela; Astilean, Simion; Vasilescu, Alina

2018-02-01

Whole cell optical biosensors, made by immobilizing whole algal, bacterial or mammalian cells on various supports have found applications in several fields, from ecology and ecotoxicity testing to biopharmaceutical production or medical diagnostics. We hereby report the deposition of functional bacterial layers of Micrococcus lysodeikticus (ML) via Matrix-Assisted Pulsed Laser Evaporation (MAPLE) on poly(diallyldimethylamonium) (PDDA)-coated-glass slides and their application as an optical biosensor for the detection of lysozyme in serum. Lysozyme is an enzyme upregulated in inflammatory diseases and ML is an enzymatic substrate for this enzyme. The MAPLE-deposited bacterial interfaces were characterised by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Fourier-Transformed Infrared Spectroscopy (FTIR), Raman and optical microscopy and were compared with control interfaces deposited via layer-by-layer on the same substrate. After MAPLE deposition and coating with graphene oxide (GO), ML-modified interfaces retained their functionality and sensitivity to lysozyme's lytic action. The optical biosensor detected lysozyme in undiluted serum in the clinically relevant range up to 10μgmL -1 , in a fast and simple manner. Copyright © 2017 Elsevier B.V. All rights reserved.

Seeing through Musculoskeletal Tissues: Improving In Situ Imaging of Bone and the Lacunar Canalicular System through Optical Clearing

PubMed Central

Berke, Ian M.; Miola, Joseph P.; David, Michael A.; Smith, Melanie K.; Price, Christopher

2016-01-01

In situ, cells of the musculoskeletal system reside within complex and often interconnected 3-D environments. Key to better understanding how 3-D tissue and cellular environments regulate musculoskeletal physiology, homeostasis, and health is the use of robust methodologies for directly visualizing cell-cell and cell-matrix architecture in situ. However, the use of standard optical imaging techniques is often of limited utility in deep imaging of intact musculoskeletal tissues due to the highly scattering nature of biological tissues. Drawing inspiration from recent developments in the deep-tissue imaging field, we describe the application of immersion based optical clearing techniques, which utilize the principle of refractive index (RI) matching between the clearing/mounting media and tissue under observation, to improve the deep, in situ imaging of musculoskeletal tissues. To date, few optical clearing techniques have been applied specifically to musculoskeletal tissues, and a systematic comparison of the clearing ability of optical clearing agents in musculoskeletal tissues has yet to be fully demonstrated. In this study we tested the ability of eight different aqueous and non-aqueous clearing agents, with RIs ranging from 1.45 to 1.56, to optically clear murine knee joints and cortical bone. We demonstrated and quantified the ability of these optical clearing agents to clear musculoskeletal tissues and improve both macro- and micro-scale imaging of musculoskeletal tissue across several imaging modalities (stereomicroscopy, spectroscopy, and one-, and two-photon confocal microscopy) and investigational techniques (dynamic bone labeling and en bloc tissue staining). Based upon these findings we believe that optical clearing, in combination with advanced imaging techniques, has the potential to complement classical musculoskeletal analysis techniques; opening the door for improved in situ investigation and quantification of musculoskeletal tissues. PMID:26930293

Seeing through Musculoskeletal Tissues: Improving In Situ Imaging of Bone and the Lacunar Canalicular System through Optical Clearing.

PubMed

Berke, Ian M; Miola, Joseph P; David, Michael A; Smith, Melanie K; Price, Christopher

2016-01-01

In situ, cells of the musculoskeletal system reside within complex and often interconnected 3-D environments. Key to better understanding how 3-D tissue and cellular environments regulate musculoskeletal physiology, homeostasis, and health is the use of robust methodologies for directly visualizing cell-cell and cell-matrix architecture in situ. However, the use of standard optical imaging techniques is often of limited utility in deep imaging of intact musculoskeletal tissues due to the highly scattering nature of biological tissues. Drawing inspiration from recent developments in the deep-tissue imaging field, we describe the application of immersion based optical clearing techniques, which utilize the principle of refractive index (RI) matching between the clearing/mounting media and tissue under observation, to improve the deep, in situ imaging of musculoskeletal tissues. To date, few optical clearing techniques have been applied specifically to musculoskeletal tissues, and a systematic comparison of the clearing ability of optical clearing agents in musculoskeletal tissues has yet to be fully demonstrated. In this study we tested the ability of eight different aqueous and non-aqueous clearing agents, with RIs ranging from 1.45 to 1.56, to optically clear murine knee joints and cortical bone. We demonstrated and quantified the ability of these optical clearing agents to clear musculoskeletal tissues and improve both macro- and micro-scale imaging of musculoskeletal tissue across several imaging modalities (stereomicroscopy, spectroscopy, and one-, and two-photon confocal microscopy) and investigational techniques (dynamic bone labeling and en bloc tissue staining). Based upon these findings we believe that optical clearing, in combination with advanced imaging techniques, has the potential to complement classical musculoskeletal analysis techniques; opening the door for improved in situ investigation and quantification of musculoskeletal tissues.

Where is crystallography going?

PubMed Central

Ashton, Alun W.; Sorensen, Thomas

2018-01-01

Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and cryo-crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments. In addition, X-ray free-electron lasers offer to capture fragments of reciprocal space without radiation damage, and open up the subpicosecond regime of protein dynamics and activity. But electrons have recently stolen the limelight: so is X-ray crystallography in rude health, or will imaging methods, especially single-particle electron microscopy, render it obsolete for the most interesting biology, whilst electron diffraction enables structure determination from even the smallest crystals? We will lay out some information to help you decide. PMID:29533241

Scabies: Advances in Noninvasive Diagnosis.

PubMed

Micali, Giuseppe; Lacarrubba, Francesco; Verzì, Anna Elisa; Chosidow, Olivier; Schwartz, Robert A

2016-06-01

Scabies is a common, highly contagious skin parasitosis caused by Sarcoptes scabiei var. hominis. Early identification and prompt treatment of infested subjects is essential, as missed diagnosis may result in outbreaks, considerable morbidity, and significantly increased economic burden. The standard diagnostic technique consists of mites' identification by microscopic examination of scales obtained by skin scraping. This is a time-consuming and risk-associated procedure that is also not suitable to a busy practice. In recent years, some advanced and noninvasive techniques such as videodermatoscopy, dermatoscopy, reflectance confocal microscopy, and optical coherence tomography have demonstrated improved efficacy in the diagnosis of scabies. Their advantages include rapid, noninvasive mass screening and post-therapeutic follow-up, with no physical risk. A greater knowledge of these techniques among general practitioners and other specialists involved in the intake care of overcrowded populations vulnerable to scabies infestations is now viewed as urgent and important in the management of outbreaks, as well as in consideration of the recent growing inflow of migrants in Europe from North Africa.

Scabies: Advances in Noninvasive Diagnosis

PubMed Central

Lacarrubba, Francesco; Verzì, Anna Elisa; Chosidow, Olivier; Schwartz, Robert A.

2016-01-01

Scabies is a common, highly contagious skin parasitosis caused by Sarcoptes scabiei var. hominis. Early identification and prompt treatment of infested subjects is essential, as missed diagnosis may result in outbreaks, considerable morbidity, and significantly increased economic burden. The standard diagnostic technique consists of mites’ identification by microscopic examination of scales obtained by skin scraping. This is a time-consuming and risk-associated procedure that is also not suitable to a busy practice. In recent years, some advanced and noninvasive techniques such as videodermatoscopy, dermatoscopy, reflectance confocal microscopy, and optical coherence tomography have demonstrated improved efficacy in the diagnosis of scabies. Their advantages include rapid, noninvasive mass screening and post-therapeutic follow-up, with no physical risk. A greater knowledge of these techniques among general practitioners and other specialists involved in the intake care of overcrowded populations vulnerable to scabies infestations is now viewed as urgent and important in the management of outbreaks, as well as in consideration of the recent growing inflow of migrants in Europe from North Africa. PMID:27311065

Advanced adaptive optics technology development

NASA Astrophysics Data System (ADS)

Olivier, Scot S.

2002-02-01

The NSF Center for Adaptive Optics (CfAO) is supporting research on advanced adaptive optics technologies. CfAO research activities include development and characterization of micro-electro-mechanical systems (MEMS) deformable mirror (DM) technology, as well as development and characterization of high-resolution adaptive optics systems using liquid crystal (LC) spatial light modulator (SLM) technology. This paper presents an overview of the CfAO advanced adaptive optics technology development activities including current status and future plans.

Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging

NASA Astrophysics Data System (ADS)

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

2015-11-01

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

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

PubMed

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

2015-11-26

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

Scanning electron microscopic characteristics of commercially available 1- and 3-piece intraocular lenses.

PubMed

Brockmann, Tobias; Brockmann, Claudia; Nietzsche, Sandor; Bertelmann, Eckart; Strobel, Juergen; Dawczynski, Jens

2013-12-01

To evaluate commercially available 1- and 3-piece intraocular lenses (IOLs) with scanning electron microscopy (SEM). Department of Ophthalmology and Electron Microscopy Center, University Hospital Jena, Jena, Germany. Experimental study. Seven +23.0 diopter IOLs of different design and material and from different manufacturers were chosen for a detailed assessment. Scanning electron microscopy was used at standardized magnifications to assess typical IOL characteristics. The particular focus was the optic edge, the optic surface, the haptic–optic junction, and the haptic. All square-edged IOLs had a curvature radius of less than 10 μm, while the mean optic edge thickness ranged between 216 μm and 382 μm. A 360-degree square-edged boundary was present in all 3-piece IOLs and in a single 1-piece model. Relevant production remnants on the optic edge were observed in 1 case. Regarding the haptic, 3-piece IOLs had uniformly shaped fibers with a mean thickness of 177 μm ± 51 (SD) (range 116 to 220 μm). Chemical adhesives were used to attach the haptic in 1 case, where alterations of the IOL material were observed. In another case, the haptic fiber was press-fitted into the optic, which resulted in bulging of the optic profile. Inspection of surface characteristics showed wavelike patterns in 2 IOLs. Taking clinical relevance into account, all IOLs were of high manufacturing quality. Certain attention was paid in creating a sharp optic edge. Surface irregularities of 2 IOLs were attributed to the manufacturing technique. Methods for implementing the haptic–optic junction were diverse.

4Pi microscopy of the nuclear pore complex.

PubMed

Kahms, Martin; Hüve, Jana; Peters, Reiner

2015-01-01

4Pi microscopy is a far-field fluorescence microscopy technique, in which the wave fronts of two opposing illuminating beams are adjusted to constructively interfere in a common focus. This yields a diffraction pattern in the direction of the optical axis, which essentially consists of a main focal spot accompanied by two smaller side lobes. At optimal conditions, the main peak of this so-called point spread function has a full width at half maximum: fixed phrase of 100 nm in the direction of the optical axis, and thus is 6-7-fold smaller than that of a confocal microscope. In this chapter, we describe the basic features of 4Pi microscopy and its application to cell biology using the example of the nuclear pore complex, a large protein assembly spanning the nuclear envelope.

Temporal focusing microscopy combined with three-dimensional structured illumination

NASA Astrophysics Data System (ADS)

Isobe, Keisuke; Toda, Keisuke; Song, Qiyuan; Kannari, Fumihiko; Kawano, Hiroyuki; Miyawaki, Atsushi; Midorikawa, Katsumi

2017-05-01

Temporal focusing microscopy provides the optical sectioning capability in wide-field two-photon fluorescence imaging. Here, we demonstrate temporal focusing microscopy combined with three-dimensional structured illumination, which enables us to enhance the three-dimensional spatial resolution and reject the background fluorescence. Experimentally, the periodic pattern of the illumination was produced not only in the lateral direction but also in the axial direction by the interference between three temporal focusing pulses, which were easily generated using a digital micromirror device. The lateral resolution and optical sectioning capability were successfully enhanced by factors of 1.6 and 3.6, respectively, compared with those of temporal focusing microscopy. In the two-photon fluorescence imaging of a tissue-like phantom, the out-of-focus background fluorescence and the scattered background fluorescence could also be rejected.

Fluorescence microscopy.

PubMed

Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

2014-10-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

Fluorescence Microscopy

PubMed Central

Sanderson, Michael J.; Smith, Ian; Parker, Ian; Bootman, Martin D.

2016-01-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. PMID:25275114

Simulated microsurgery monitoring using intraoperative multimodal surgical microscopy

NASA Astrophysics Data System (ADS)

Lee, Donghyun; Lee, Changho; Kim, Sehui; Zhou, Qifa; Kim, Jeehyun; Kim, Chulhong

2016-03-01

We have developed an intraoperative multimodal surgical microscopy system that provides simultaneous real-time enlarged surface views and subsurface anatomic information during surgeries by integrating spectral domain optical coherence tomography (SD-OCT), optical-resolution photoacoustic microscopy (OR-PAM), and conventional surgical microscopy. By sharing the same optical path, both OCT and PAM images were simultaneously acquired. Additionally, the custom-made needle-type transducer received the generated PA signals enabling convenient surgical operation without using a water bath. Using a simple augmented device, the OCT and PAM images were projected on the view plane of the surgical microscope. To quantify the performance of our system, we measured spatial resolutions of our system. Then, three microsurgery simulation and analysis were processed: (1) ex vivo needle tracking and monitoring injection of carbon particles in biological tissues, (2) in vivo needle tracking and monitoring injection of carbon particles in tumor-bearing mice, and (3) in vivo guiding of melanoma removal in melanoma-bearing mice. The results indicate that this triple modal system is useful for intraoperative purposes, and can potentially be a vital tool in microsurgeries.

Quantitative DIC microscopy using an off-axis self-interference approach.

PubMed

Fu, Dan; Oh, Seungeun; Choi, Wonshik; Yamauchi, Toyohiko; Dorn, August; Yaqoob, Zahid; Dasari, Ramachandra R; Feld, Michael S

2010-07-15

Traditional Normarski differential interference contrast (DIC) microscopy is a very powerful method for imaging nonstained biological samples. However, one of its major limitations is the nonquantitative nature of the imaging. To overcome this problem, we developed a quantitative DIC microscopy method based on off-axis sample self-interference. The digital holography algorithm is applied to obtain quantitative phase gradients in orthogonal directions, which leads to a quantitative phase image through a spiral integration of the phase gradients. This method is practically simple to implement on any standard microscope without stringent requirements on polarization optics. Optical sectioning can be obtained through enlarged illumination NA.

Super resolution imaging of HER2 gene amplification

NASA Astrophysics Data System (ADS)

Okada, Masaya; Kubo, Takuya; Masumoto, Kanako; Iwanaga, Shigeki

2016-02-01

HER2 positive breast cancer is currently examined by counting HER2 genes using fluorescence in situ hybridization (FISH)-stained breast carcinoma samples. In this research, two-dimensional super resolution fluorescence microscopy based on stochastic optical reconstruction microscopy (STORM), with a spatial resolution of approximately 20 nm in the lateral direction, was used to more precisely distinguish and count HER2 genes in a FISH-stained tissue section. Furthermore, by introducing double-helix point spread function (DH-PSF), an optical phase modulation technique, to super resolution microscopy, three-dimensional images were obtained of HER2 in a breast carcinoma sample approximately 4 μm thick.

Biological applications of near-field scanning optical microscopy

NASA Astrophysics Data System (ADS)

Moers, Marco H. P.; Ruiter, A. G. T.; Jalocha, Alain; van Hulst, Niko F.; Kalle, W. H. J.; Wiegant, J. C. A. G.; Raap, A. K.

1995-09-01

Near-field Scanning Optical Microscopy (NSOM) is a true optical microscopic technique allowing fluorescence, absorption, reflection and polarization contrast with the additional advantage of nanometer lateral resolution, unlimited by diffraction and operation at ambient conditions. NSOM based on metal coated adiabatically tapered fibers, combined with shear force feedback and operated in illumination mode, has proven to be the most powerful NSOM arrangement, because of its true localization of the optical interaction, its various optical contrast possibilities and its sensitivity down to the single molecular level. In this paper applications of `aperture' NSOM to Fluorescence In Situ Hybridization of human metaphase chromosomes are presented, where the localized fluorescence allows to identify specific DNA sequences. All images are accompanied by the simultaneously acquired force image, enabling direct comparison of the optical contrast with the sample topography on nanometer scale, far beyond the diffraction limit. Thus the unique combination of high resolution, specific optical contrast and ambient operation offers many new direction possibilities in biological studies.

A review of cellphone microscopy for disease detection.

PubMed

Dendere, R; Myburg, N; Douglas, T S

2015-12-01

The expansion in global cellphone network coverage coupled with advances in cellphone imaging capabilities present an opportunity for the advancement of cellphone microscopy as a low-cost alternative to conventional microscopy for disease detection in resource-limited regions. The development of cellphone microscopy has also benefitted from the availability of low-cost miniature microscope components such as low-power light-emitting diodes and ball lenses. As a result, researchers are developing hardware and software techniques that would enable such microscopes to produce high-resolution, diagnostic-quality images. This approach may lead to more widespread delivery of diagnostic services in resource-limited areas where there is a shortage of the skilled labour required for conventional microscopy and where prevalence of infectious and other diseases is still high. In this paper, we review current techniques, clinical applications and challenges faced in the field of cellphone microscopy. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.