In situ flat embedding of monolayers and cell relocation in the acrylic resin LR white for comparative light and electron microscopy studies.

PubMed

Steiner, M; Schöfer, C; Mosgoeller, W

1994-12-01

A simple and reliable method has been developed for the in situ LR White embedding of cell monolayers grown on glass cover-slips. Combined with cytochemical or immunological procedures, this technique allows light and/or electron microscopy investigations of a large number of cells in the same horizontal plane within a relatively short period of time. It can be applied to cells grown on microgrid finder cover-slips which allows a distinct site of even an individual cell of a monolayer to be studied at first at the light microscope level and subsequently at the electron microscope level. Hence, it is also suitable for controlling manipulation of single cells, followed by their serial sectioning after relocation in the electron microscope.

Adaptive optics plug-and-play setup for high-resolution microscopes with multi-actuator adaptive lens

NASA Astrophysics Data System (ADS)

Quintavalla, M.; Pozzi, P.; Verhaegen, Michelle; Bijlsma, Hielke; Verstraete, Hans; Bonora, S.

2018-02-01

Adaptive Optics (AO) has revealed as a very promising technique for high-resolution microscopy, where the presence of optical aberrations can easily compromise the image quality. Typical AO systems however, are almost impossible to implement on commercial microscopes. We propose a simple approach by using a Multi-actuator Adaptive Lens (MAL) that can be inserted right after the objective and works in conjunction with an image optimization software allowing for a wavefront sensorless correction. We presented the results obtained on several commercial microscopes among which a confocal microscope, a fluorescence microscope, a light sheet microscope and a multiphoton microscope.

Advanced imaging techniques II: using a compound microscope for photographing point-mount specimens

USDA-ARS?s Scientific Manuscript database

Digital imaging technology has revolutionized the practice photographing insects for scientific study. Herein described are lighting and mounting techniques designed for imaging micro Hymenoptera. Techniques described here are applicable to all small insects, as well as other invertebrates. The ke...

Volumetric bioimaging based on light field microscopy with temporal focusing illumination

NASA Astrophysics Data System (ADS)

Hsu, Feng-Chun; Sie, Yong Da; Lai, Feng-Jie; Chen, Shean-Jen

2018-02-01

Light field technique at a single shot can get the whole volume image of observed sample. Therefore, the original frame rate of the optical system can be taken as the volumetric image rate. For dynamically imaging whole micron-scale biosample, a light field microscope with temporal focusing illumination has been developed. In the light field microscope, the f-number of the microlens array (MLA) is adopted to match that of the objective; hence, the subimages via adjacent lenslets do not overlay each other. A three-dimensional (3D) deconvolution algorithm is utilized to deblur the out-of-focusing part. Conventional light field microscopy (LFM) illuminates whole volume sample even noninteresting parts; nevertheless, whole volume excitation causes even more damage on bio-sample and also increase the background noise from the out of range. Therefore, temporal focusing is integrated into the light field microscope for selecting the illumination volume. Herein, a slit on the back focal plane of the objective is utilized to control the axial excitation confinement for selecting the illumination volume. As a result, the developed light field microscope with the temporal focusing multiphoton illumination (TFMPI) can reconstruct 3D images within the selected volume, and the lateral resolution approaches to the theoretical value. Furthermore, the 3D Brownian motion of two-micron fluorescent beads is observed as the criterion of dynamic sample. With superior signal-to-noise ratio and less damage to tissue, the microscope is potential to provide volumetric imaging for vivo sample.

A multi-modal stereo microscope based on a spatial light modulator.

PubMed

Lee, M P; Gibson, G M; Bowman, R; Bernet, S; Ritsch-Marte, M; Phillips, D B; Padgett, M J

2013-07-15

Spatial Light Modulators (SLMs) can emulate the classic microscopy techniques, including differential interference (DIC) contrast and (spiral) phase contrast. Their programmability entails the benefit of flexibility or the option to multiplex images, for single-shot quantitative imaging or for simultaneous multi-plane imaging (depth-of-field multiplexing). We report the development of a microscope sharing many of the previously demonstrated capabilities, within a holographic implementation of a stereo microscope. Furthermore, we use the SLM to combine stereo microscopy with a refocusing filter and with a darkfield filter. The instrument is built around a custom inverted microscope and equipped with an SLM which gives various imaging modes laterally displaced on the same camera chip. In addition, there is a wide angle camera for visualisation of a larger region of the sample.

Three-dimensional microscopic tomographic imagings of the cataract in a human lens in vivo

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1998-10-01

The problem of three-dimensional visualization of a human lens in vivo has been solved by a technique of volume rendering a transformed series of 60 rotated Scheimpflug (a dual slit reflected light microscope) digital images. The data set was obtained by rotating the Scheimpflug camera about the optic axis of the lens in 3 degree increments. The transformed set of optical sections were first aligned to correct for small eye movements, and then rendered into a volume reconstruction with volume rendering computer graphics techniques. To help visualize the distribution of lens opacities (cataracts) in the living, human lens the intensity of light scattering was pseudocolor coded and the cataract opacities were displayed as a movie.

Resolution enhancement techniques in microscopy

NASA Astrophysics Data System (ADS)

Cremer, Christoph; Masters, Barry R.

2013-05-01

We survey the history of resolution enhancement techniques in microscopy and their impact on current research in biomedicine. Often these techniques are labeled superresolution, or enhanced resolution microscopy, or light-optical nanoscopy. First, we introduce the development of diffraction theory in its relation to enhanced resolution; then we explore the foundations of resolution as expounded by the astronomers and the physicists and describe the conditions for which they apply. Then we elucidate Ernst Abbe's theory of optical formation in the microscope, and its experimental verification and dissemination to the world wide microscope communities. Second, we describe and compare the early techniques that can enhance the resolution of the microscope. Third, we present the historical development of various techniques that substantially enhance the optical resolution of the light microscope. These enhanced resolution techniques in their modern form constitute an active area of research with seminal applications in biology and medicine. Our historical survey of the field of resolution enhancement uncovers many examples of reinvention, rediscovery, and independent invention and development of similar proposals, concepts, techniques, and instruments. Attribution of credit is therefore confounded by the fact that for understandable reasons authors stress the achievements from their own research groups and sometimes obfuscate their contributions and the prior art of others. In some cases, attribution of credit is also made more complex by the fact that long term developments are difficult to allocate to a specific individual because of the many mutual connections often existing between sometimes fiercely competing, sometimes strongly collaborating groups. Since applications in biology and medicine have been a major driving force in the development of resolution enhancing approaches, we focus on the contribution of enhanced resolution to these fields.

Space-resolved diffusing wave spectroscopy measurements of the macroscopic deformation and the microscopic dynamics in tensile strain tests

NASA Astrophysics Data System (ADS)

Nagazi, Med-Yassine; Brambilla, Giovanni; Meunier, Gérard; Marguerès, Philippe; Périé, Jean-Noël; Cipelletti, Luca

2017-01-01

We couple a laser-based, space-resolved dynamic light scattering apparatus to a universal traction machine for mechanical extensional tests. We perform simultaneous optical and mechanical measurements on polyether ether ketone, a semi-crystalline polymer widely used in the industry. Due to the high turbidity of the sample, light is multiply scattered by the sample and the diffusing wave spectroscopy (DWS) formalism is used to interpret the data. Space-resolved DWS yields spatial maps of the sample strain and of the microscopic dynamics. An excellent agreement is found between the strain maps thus obtained and those measured by a conventional stereo-digital image correlation technique. The microscopic dynamics reveals both affine motion and plastic rearrangements. Thanks to the extreme sensitivity of DWS to displacements as small as 1 nm, plastic activity and its spatial localization can be detected at an early stage of the sample strain, making the technique presented here a valuable complement to existing material characterization methods.

Phase Sensitive Demodulation in Multiphoton Microscopy

NASA Astrophysics Data System (ADS)

Fisher, Walt G.; Piston, David W.; Wachter, Eric A.

2002-06-01

Multiphoton laser scanning microscopy offers advantages in depth of penetration into intact samples over other optical sectioning techniques. To achieve these advantages it is necessary to detect the emitted light without spatial filtering. In this nondescanned (nonconfocal) approach, ambient room light can easily contaminate the signal, forcing experiments to be performed in absolute darkness. For multiphoton microscope systems employing mode-locked lasers, signal processing can be used to reduce such problems by taking advantage of the pulsed characteristics of such lasers. Specifically, by recovering fluorescence generated at the mode-locked frequency, interference from stray light and other ambient noise sources can be significantly reduced. This technology can be adapted to existing microscopes by inserting demodulation circuitry between the detector and data collection system. The improvement in signal-to-noise ratio afforded by this approach yields a more robust microscope system and opens the possibility of moving multiphoton microscopy from the research lab to more demanding settings, such as the clinic.

A simple water-immersion condenser for imaging living brain slices on an inverted microscope.

PubMed

Prusky, G T

1997-09-05

Due to some physical limitations of conventional condensers, inverted compound microscopes are not optimally suited for imaging living brain slices with transmitted light. Herein is described a simple device that converts an inverted microscope into an effective tool for this application by utilizing an objective as a condenser. The device is mounted on a microscope in place of the condenser, is threaded to accept a water immersion objective, and has a slot for a differential interference contrast (DIC) slider. When combined with infrared video techniques, this device allows an inverted microscope to effectively image living cells within thick brain slices in an open perfusion chamber.

A compact light-sheet microscope for the study of the mammalian central nervous system

PubMed Central

Yang, Zhengyi; Haslehurst, Peter; Scott, Suzanne; Emptage, Nigel; Dholakia, Kishan

2016-01-01

Investigation of the transient processes integral to neuronal function demands rapid and high-resolution imaging techniques over a large field of view, which cannot be achieved with conventional scanning microscopes. Here we describe a compact light sheet fluorescence microscope, featuring a 45° inverted geometry and an integrated photolysis laser, that is optimized for applications in neuroscience, in particular fast imaging of sub-neuronal structures in mammalian brain slices. We demonstrate the utility of this design for three-dimensional morphological reconstruction, activation of a single synapse with localized photolysis, and fast imaging of neuronal Ca2+ signalling across a large field of view. The developed system opens up a host of novel applications for the neuroscience community. PMID:27215692

Remote microscopy and volumetric imaging on the surface of icy satellites

NASA Astrophysics Data System (ADS)

Soto, Alejandro; Nowicki, Keith; Howett, Carly; Feldkhun, Daniel; Retherford, Kurt D.

2017-10-01

With NASA PIDDP support we have applied recent advancements in Fourier-domain microscopy to develop an instrument capable of microscopic imaging from meter-scale distances for use on a planetary lander on the surface of an icy satellite or other planetary bodies. Without moving parts, our instrument projects dynamic patterns of laser light onto a distant target using a lightweight large-aperture reflector, which then collects the light scattered or fluoresced by the target on a fast photon-bucket detector. Using Fourier Transform based techniques, we reconstruct an image from the detected light. The remote microscope has been demonstrated to produce 2D images with better than 15 micron lateral resolution for targets at a distance of 5 meters and is capable of linearly proportionally higher resolution at shorter distances. The remote microscope is also capable of providing three-dimensional (3D) microscopic imaging capabilities, allowing future surface scientists to explore the morphology of microscopic features in surface ices, for example. The instrument enables microscopic in-situ imaging during day or night without the use of a robotic arm, greatly facilitating the surface operations for a lander or rover while expanding the area of investigation near a landing site for improved science targeting. We are developing this remote microscope for in-situ planetary exploration as a collaboration between the Southwest Research Institute, LambdaMetrics, and the University of Colorado.

Sensing of Streptococcus mutans by microscopic imaging ellipsometry

NASA Astrophysics Data System (ADS)

Khaleel, Mai Ibrahim; Chen, Yu-Da; Chien, Ching-Hang; Chang, Yia-Chung

2017-05-01

Microscopic imaging ellipsometry is an optical technique that uses an objective and sensing procedure to measure the ellipsometric parameters Ψ and Δ in the form of microscopic maps. This technique is well known for being noninvasive and label-free. Therefore, it can be used to detect and characterize biological species without any impact. Microscopic imaging ellipsometry was used to measure the optical response of dried Streptococcus mutans cells on a glass substrate. The ellipsometric Ψ and Δ maps were obtained with the Optrel Multiskop system for specular reflection in the visible range (λ=450 to 750 nm). The Ψ and Δ images at 500, 600, and 700 nm were analyzed using three different theoretical models with single-bounce, two-bounce, and multibounce light paths to obtain the optical constants and height distribution. The obtained images of the optical constants show different aspects when comparing the single-bounce analysis with the two-bounce or multibounce analysis in detecting S. mutans samples. Furthermore, the height distributions estimated by two-bounce and multibounce analyses of S. mutans samples were in agreement with the thickness values measured by AFM, which implies that the two-bounce and multibounce analyses can provide information complementary to that obtained by a single-bounce light path.

Analysis of thick brain sections by obverse-reverse computer microscopy: application of a new, high clarity Golgi-Nissl stain.

PubMed

Glaser, E M; Van der Loos, H

1981-08-01

Exceptionally clear Golgi-Nissl sections of 300 micron thickness have been morphometrically studied by light microscopy using oil immersion objectives. The clarity results from a new variation of a staining procedure that combines Golgi and Nissl images in one section. A viewing technique has been developed that permits a histologic preparation to be examined from its obverse (or normally viewed) side and its reverse (or under) side. The technique was designed for use with a computer microscope but can be employed with any light microscope whose stage position can be measured within 100 micron. Sections thicker than 300 micron can be studied dependent on the working distance of the objective lens, provided that the clarity of the material permits it.

The microscopes of Antoni van Leeuwenhoek.

PubMed

van Zuylen, J

1981-03-01

The seventeenth-century Dutch microscopist, Antoni van Leeuwenhoek, was the first man to make a protracted study of microscopical objects, and, unlike his contemporary Robert Hooke, he viewed by transmitted light. Leeuwenhoek made over 500 of his own, curious, simple microscopes, but now only nine are known to exist. The exact nature of the lenses Leeuwenhoek made, has for long been a puzzle. The existing microscopes have now been examined in detail, and their optical characteristics measured and tabulated. It is proposed that the lens of highest magnification, x 266, was made using a special blown bubble technique.

Microstructural study of the nickel-base alloy WAZ-20 using qualitative and quantitative electron optical techniques

NASA Technical Reports Server (NTRS)

Young, S. G.

1973-01-01

The NASA nickel-base alloy WAZ-20 was analyzed by advanced metallographic techniques to qualitatively and quantitatively characterize its phases and stability. The as-cast alloy contained primary gamma-prime, a coarse gamma-gamma prime eutectic, a gamma-fine gamma prime matrix, and MC carbides. A specimen aged at 870 C for 1000 hours contained these same constituents and a few widely scattered high W particles. No detrimental phases (such as sigma or mu) were observed. Scanning electron microscope, light metallography, and replica electron microscope methods are compared. The value of quantitative electron microprobe techniques such as spot and area analysis is demonstrated.

Fractal propagation method enables realistic optical microscopy simulations in biological tissues

PubMed Central

Glaser, Adam K.; Chen, Ye; Liu, Jonathan T.C.

2017-01-01

Current simulation methods for light transport in biological media have limited efficiency and realism when applied to three-dimensional microscopic light transport in biological tissues with refractive heterogeneities. We describe here a technique which combines a beam propagation method valid for modeling light transport in media with weak variations in refractive index, with a fractal model of refractive index turbulence. In contrast to standard simulation methods, this fractal propagation method (FPM) is able to accurately and efficiently simulate the diffraction effects of focused beams, as well as the microscopic heterogeneities present in tissue that result in scattering, refractive beam steering, and the aberration of beam foci. We validate the technique and the relationship between the FPM model parameters and conventional optical parameters used to describe tissues, and also demonstrate the method’s flexibility and robustness by examining the steering and distortion of Gaussian and Bessel beams in tissue with comparison to experimental data. We show that the FPM has utility for the accurate investigation and optimization of optical microscopy methods such as light-sheet, confocal, and nonlinear microscopy. PMID:28983499

eduSPIM: Light Sheet Microscopy in the Museum

PubMed Central

Schmid, Benjamin; Weber, Michael; Huisken, Jan

2016-01-01

Light Sheet Microscopy in the Museum Light sheet microscopy (or selective plane illumination microscopy) is an important imaging technique in the life sciences. At the same time, this technique is also ideally suited for community outreach projects, because it produces visually appealing, highly dynamic images of living organisms and its working principle can be understood with basic optics knowledge. Still, the underlying concepts are widely unknown to the non-scientific public. On the occasion of the UNESCO International Year of Light, a technical museum in Dresden, Germany, launched a special, interactive exhibition. We built a fully functional, educational selective plane illumination microscope (eduSPIM) to demonstrate how developments in microscopy promote discoveries in biology. Design Principles of an Educational Light Sheet Microscope To maximize educational impact, we radically reduced a standard light sheet microscope to its essential components without compromising functionality and incorporated stringent safety concepts beyond those needed in the lab. Our eduSPIM system features one illumination and one detection path and a sealed sample chamber. We image fixed zebrafish embryos with fluorescent vasculature, because the structure is meaningful to laymen and visualises the optical principles of light sheet microscopy. Via a simplified interface, visitors acquire fluorescence and transmission data simultaneously. The eduSPIM Design Is Tailored Easily to Fit Numerous Applications The universal concepts presented here may also apply to other scientific approaches that are communicated to laymen in interactive settings. The specific eduSPIM design is adapted easily for various outreach and teaching activities. eduSPIM may even prove useful for labs needing a simple SPIM. A detailed parts list and schematics to rebuild eduSPIM are provided. PMID:27560188

Total internal reflection and dynamic light scattering microscopy of gels

NASA Astrophysics Data System (ADS)

Gregor, Brian F.

Two different techniques which apply optical microscopy in novel ways to the study of biological systems and materials were built and applied to several samples. The first is a system for adapting the well-known technique of dynamic light scattering (DLS) to an optical microscope. This can detect and scatter light from very small volumes, as compared to standard DLS which studies light scattering from volumes 1000x larger. The small scattering volume also allows for the observation of nonergodic dynamics in appropriate samples. Porcine gastric mucin (PGM) forms a gel at low pH which lines the epithelial cell layer and acts as a protective barrier against the acidic stomach environment. The dynamics and microscopic viscosity of PGM at different pH levels is studied using polystyrene microspheres as tracer particles. The microscopic viscosity and microrheological properties of the commercial basement membrane Matrigel are also studied with this instrument. Matrigel is frequently used to culture cells and its properties remain poorly determined. Well-characterized and purely synthetic Matrigel substitutes will need to have the correct rheological and morphological characteristics. The second instrument designed and built is a microscope which uses an interferometry technique to achieve an improvement in resolution 2.5x better in one dimension than the Abbe diffraction limit. The technique is based upon the interference of the evanescent field generated on the surface of a prism by a laser in a total internal reflection geometry. The enhanced resolution is demonstrated with fluorescent samples. Additionally. Raman imaging microscopy is demonstrated using the evanescent field in resonant and non-resonant samples, although attempts at applying the enhanced resolution technique to the Raman images were ultimately unsuccessful. Applications of this instrument include high resolution imaging of cell membranes and macroscopic structures in gels and proteins. Finally, a third section incorporating previous research on simulations of complex fluids is included. Two dimensional simulations of oil, water, and surfactant mixtures were computed with a lattice gas method. The simulated systems were randomly mixed and then the temperature was quenched to a predetermined point. Spontaneous micellization is observed for a narrow range of temperature quenches, and the overall growth rate of macroscopic structure is found to follow a Vogel-Fulcher growth law.

Superresolution Imaging with Standard Fluorescent Probes

PubMed Central

Burnette, Dylan T.; Lippincott-Schwartz, Jennifer; Kachar, Bechara

2013-01-01

For more than 100 years, the ultimate resolution of a light microscope (~200 nm) has been constrained by the fundamental physical phenomenon of diffraction, as described by Ernst Abbe in 1873. While this limitation is just as applicable to today’s light microscopes, it is the combination of high-end optics, clever methods of sample illumination, and computational techniques that has enabled researchers to access high-resolution information an order of magnitude greater than once thought possible. This combination, broadly termed superresolution microscopy, has been increasingly practical for many labs to implement from both a hardware and software standpoint, but as with many cutting-edge techniques, it also comes with limitations. One of the current drawbacks to superresolution microscopy is the limited number of probes and conditions that have been suitable for imaging. Here, a technique termed bleaching/blinking assisted localization microscopy (BaLM) makes use of almost all fluorophore’s inherent blinking and bleaching properties as a means to generate superresolution images. PMID:24510788

Enhanced optical coupling and Raman scattering via microscopic interface engineering

NASA Astrophysics Data System (ADS)

Thompson, Jonathan V.; Hokr, Brett H.; Kim, Wihan; Ballmann, Charles W.; Applegate, Brian E.; Jo, Javier A.; Yamilov, Alexey; Cao, Hui; Scully, Marlan O.; Yakovlev, Vladislav V.

2017-11-01

Spontaneous Raman scattering is an extremely powerful tool for the remote detection and identification of various chemical materials. However, when those materials are contained within strongly scattering or turbid media, as is the case in many biological and security related systems, the sensitivity and range of Raman signal generation and detection is severely limited. Here, we demonstrate that through microscopic engineering of the optical interface, the optical coupling of light into a turbid material can be substantially enhanced. This improved coupling facilitates the enhancement of the Raman scattering signal generated by molecules within the medium. In particular, we detect at least two-orders of magnitude more spontaneous Raman scattering from a sample when the pump laser light is focused into a microscopic hole in the surface of the sample. Because this approach enhances both the interaction time and interaction region of the laser light within the material, its use will greatly improve the range and sensitivity of many spectroscopic techniques, including Raman scattering and fluorescence emission detection, inside highly scattering environments.

Applied physics: Optical trapping for space mirrors.

PubMed

McGloin, David

2014-02-27

Might it be possible to create mirrors for space telescopes, using nothing but microscopic particles held in place by light? A study that exploits a technique called optical binding provides a step towards this goal.

Dual-mode optical microscope based on single-pixel imaging

NASA Astrophysics Data System (ADS)

Rodríguez, A. D.; Clemente, P.; Tajahuerce, E.; Lancis, J.

2016-07-01

We demonstrate an inverted microscope that can image specimens in both reflection and transmission modes simultaneously with a single light source. The microscope utilizes a digital micromirror device (DMD) for patterned illumination altogether with two single-pixel photosensors for efficient light detection. The system, a scan-less device with no moving parts, works by sequential projection of a set of binary intensity patterns onto the sample that are codified onto a modified commercial DMD. Data to be displayed are geometrically transformed before written into a memory cell to cancel optical artifacts coming from the diamond-like shaped structure of the micromirror array. The 24-bit color depth of the display is fully exploited to increase the frame rate by a factor of 24, which makes the technique practicable for real samples. Our commercial DMD-based LED-illumination is cost effective and can be easily coupled as an add-on module for already existing inverted microscopes. The reflection and transmission information provided by our dual microscope complement each other and can be useful for imaging non-uniform samples and to prevent self-shadowing effects.

Scanning thin-sheet laser imaging microscopy (sTSLIM) with structured illumination and HiLo background rejection.

PubMed Central

Schröter, Tobias J.; Johnson, Shane B.; John, Kerstin; Santi, Peter A.

2011-01-01

We report replacement of one side of a static illumination, dual sided, thin-sheet laser imaging microscope (TSLIM) with an intensity modulated laser scanner in order to implement structured illumination (SI) and HiLo image demodulation techniques for background rejection. The new system is equipped with one static and one scanned light-sheet and is called a scanning thin-sheet laser imaging microscope (sTSLIM). It is an optimized version of a light-sheet fluorescent microscope that is designed to image large specimens (<15 mm in diameter). In this paper we describe the hardware and software modifications to TSLIM that allow for static and uniform light-sheet illumination with SI and HiLo image demodulation. The static light-sheet has a thickness of 3.2 µm; whereas, the scanned side has a light-sheet thickness of 4.2 µm. The scanned side images specimens with subcellular resolution (<1 µm lateral and <4 µm axial resolution) with a size up to 15 mm. SI and HiLo produce superior contrast compared to both the uniform static and scanned light-sheets. HiLo contrast was greater than SI and is faster and more robust than SI because as it produces images in two-thirds of the time and exhibits fewer intensity streaking artifacts. PMID:22254177

Micro axial tomography: A miniaturized, versatile stage device to overcome resolution anisotropy in fluorescence light microscopy

NASA Astrophysics Data System (ADS)

Staier, Florian; Eipel, Heinz; Matula, Petr; Evsikov, Alexei V.; Kozubek, Michal; Cremer, Christoph; Hausmann, Michael

2011-09-01

With the development of novel fluorescence techniques, high resolution light microscopy has become a challenging technique for investigations of the three-dimensional (3D) micro-cosmos in cells and sub-cellular components. So far, all fluorescence microscopes applied for 3D imaging in biosciences show a spatially anisotropic point spread function resulting in an anisotropic optical resolution or point localization precision. To overcome this shortcoming, micro axial tomography was suggested which allows object tilting on the microscopic stage and leads to an improvement in localization precision and spatial resolution. Here, we present a miniaturized device which can be implemented in a motor driven microscope stage. The footprint of this device corresponds to a standard microscope slide. A special glass fiber can manually be adjusted in the object space of the microscope lens. A stepwise fiber rotation can be controlled by a miniaturized stepping motor incorporated into the device. By means of a special mounting device, test particles were fixed onto glass fibers, optically localized with high precision, and automatically rotated to obtain views from different perspective angles under which distances of corresponding pairs of objects were determined. From these angle dependent distance values, the real 3D distance was calculated with a precision in the ten nanometer range (corresponding here to an optical resolution of 10-30 nm) using standard microscopic equipment. As a proof of concept, the spindle apparatus of a mature mouse oocyte was imaged during metaphase II meiotic arrest under different perspectives. Only very few images registered under different rotation angles are sufficient for full 3D reconstruction. The results indicate the principal advantage of the micro axial tomography approach for many microscopic setups therein and also those of improved resolutions as obtained by high precision localization determination.

Modular low-light microscope for imaging cellular bioluminescence and radioluminescence

PubMed Central

Kim, Tae Jin; Türkcan, Silvan; Pratx, Guillem

2017-01-01

Low-light microscopy methods are receiving increased attention as new applications have emerged. One such application is to allow longitudinal imaging of light-sensitive cells with no phototoxicity and no photobleaching of fluorescent biomarkers. Another application is for imaging signals that are inherently dim and undetectable using standard microscopy, such as bioluminescence, chemiluminescence, or radioluminescence. In this protocol, we provide instructions on how to build a modular low-light microscope (1-4 d) by coupling two microscope objective lenses, back-to-back from each other, using standard optomechanical components. We also provide directions on how to image dim signals such as radioluminescence (1-1.5 h), bioluminescence (∼30 min) and low-excitation fluorescence (∼15 min). In particular, radioluminescence microscopy is explained in detail as it is a newly developed technique, which enables the study of small molecule transport (eg. radiolabeled drugs, metabolic precursors, and nuclear medicine contrast agents) by single cells without perturbing endogenous biochemical processes. In this imaging technique, a scintillator crystal (eg. CdWO4) is placed in close proximity to the radiolabeled cells, where it converts the radioactive decays into optical flashes detectable using a sensitive camera. Using the image reconstruction toolkit provided in this protocol, the flashes can be reconstructed to yield high-resolution image of the radiotracer distribution. With appropriate timing, the three aforementioned imaging modalities may be performed altogether on a population of live cells, allowing the user to perform parallel functional studies of cell heterogeneity at the single-cell level. PMID:28426025

Sheet-scanned dual-axis confocal microscopy using Richardson-Lucy deconvolution.

PubMed

Wang, D; Meza, D; Wang, Y; Gao, L; Liu, J T C

2014-09-15

We have previously developed a line-scanned dual-axis confocal (LS-DAC) microscope with subcellular resolution suitable for high-frame-rate diagnostic imaging at shallow depths. Due to the loss of confocality along one dimension, the contrast (signal-to-background ratio) of a LS-DAC microscope is deteriorated compared to a point-scanned DAC microscope. However, by using a sCMOS camera for detection, a short oblique light-sheet is imaged at each scanned position. Therefore, by scanning the light sheet in only one dimension, a thin 3D volume is imaged. Both sequential two-dimensional deconvolution and three-dimensional deconvolution are performed on the thin image volume to improve the resolution and contrast of one en face confocal image section at the center of the volume, a technique we call sheet-scanned dual-axis confocal (SS-DAC) microscopy.

Use of a night vision intensifier for direct visualization by eye of far-red and near-infrared fluorescence through an optical microscope.

PubMed

Siddiqi, M A; Kilduff, G M; Gearhart, J D

2003-11-01

We describe the design, construction and testing of a prototype device that allows the direct visualization by eye of far-red and near-infrared (NIR) fluorescence through an optical microscope. The device incorporates a gallium arsenide (GaAs) image intensifier, typically utilized in low-light or 'night vision' applications. The intensifier converts far-red and NIR light into electrons and then into green light, which is visible to the human eye. The prototype makes possible the direct, real-time viewing by eye of normally invisible far-red and NIR fluorescence from a wide variety of fluorophores, using the full field of view of the microscope to which it is applied. The high sensitivity of the image intensifier facilitates the viewing of a wide variety of photosensitive specimens, including live cells and embryos, at vastly reduced illumination levels in both fluorescence and bright-field microscopy. Modifications to the microscope are not required in order to use the prototype, which is fully compatible with all current fluorescence techniques. Refined versions of the prototype device will have broad research and clinical applications.

Bessel light sheet structured illumination microscopy

NASA Astrophysics Data System (ADS)

Noshirvani Allahabadi, Golchehr

Biomedical study researchers using animals to model disease and treatment need fast, deep, noninvasive, and inexpensive multi-channel imaging methods. Traditional fluorescence microscopy meets those criteria to an extent. Specifically, two-photon and confocal microscopy, the two most commonly used methods, are limited in penetration depth, cost, resolution, and field of view. In addition, two-photon microscopy has limited ability in multi-channel imaging. Light sheet microscopy, a fast developing 3D fluorescence imaging method, offers attractive advantages over traditional two-photon and confocal microscopy. Light sheet microscopy is much more applicable for in vivo 3D time-lapsed imaging, owing to its selective illumination of tissue layer, superior speed, low light exposure, high penetration depth, and low levels of photobleaching. However, standard light sheet microscopy using Gaussian beam excitation has two main disadvantages: 1) the field of view (FOV) of light sheet microscopy is limited by the depth of focus of the Gaussian beam. 2) Light-sheet images can be degraded by scattering, which limits the penetration of the excitation beam and blurs emission images in deep tissue layers. While two-sided sheet illumination, which doubles the field of view by illuminating the sample from opposite sides, offers a potential solution, the technique adds complexity and cost to the imaging system. We investigate a new technique to address these limitations: Bessel light sheet microscopy in combination with incoherent nonlinear Structured Illumination Microscopy (SIM). Results demonstrate that, at visible wavelengths, Bessel excitation penetrates up to 250 microns deep in the scattering media with single-side illumination. Bessel light sheet microscope achieves confocal level resolution at a lateral resolution of 0.3 micron and an axial resolution of 1 micron. Incoherent nonlinear SIM further reduces the diffused background in Bessel light sheet images, resulting in confocal quality images in thick tissue. The technique was applied to live transgenic zebra fish tg(kdrl:GFP), and the sub-cellular structure of fish vasculature genetically labeled with GFP was captured in 3D. The superior speed of the microscope enables us to acquire signal from 200 layers of a thick sample in 4 minutes. The compact microscope uses exclusively off-the-shelf components and offers a low-cost imaging solution for studying small animal models or tissue samples.

Optical forces, torques, and force densities calculated at a microscopic level using a self-consistent hydrodynamics method

NASA Astrophysics Data System (ADS)

Ding, Kun; Chan, C. T.

2018-04-01

The calculation of optical force density distribution inside a material is challenging at the nanoscale, where quantum and nonlocal effects emerge and macroscopic parameters such as permittivity become ill-defined. We demonstrate that the microscopic optical force density of nanoplasmonic systems can be defined and calculated using the microscopic fields generated using a self-consistent hydrodynamics model that includes quantum, nonlocal, and retardation effects. We demonstrate this technique by calculating the microscopic optical force density distributions and the optical binding force induced by external light on nanoplasmonic dimers. This approach works even in the limit when the nanoparticles are close enough to each other so that electron tunneling occurs, a regime in which classical electromagnetic approach fails completely. We discover that an uneven distribution of optical force density can lead to a light-induced spinning torque acting on individual particles. The hydrodynamics method offers us an accurate and efficient approach to study optomechanical behavior for plasmonic systems at the nanoscale.

Application of microscopy in authentication of traditional Tibetan medicinal plants of five Rhodiola (Crassulaceae) alpine species by comparative anatomy and micromorphology.

PubMed

Li, Tao; Zhang, Hao

2008-06-01

A comparative analysis was undertaken to conduct an anatomical and micromorphological study of five species of Rhodiola-R. kirilowii, R. yunnanensis, R. crenulata, R. fastigata, and R. quadrifida-collected from the western Sichuan province plateau of China. Rhodiola plants are a popularly used ethnodrug from the Qinghai-Tibetan plateau of China. Modern studies have shown that the plants of Rhodiola possess different pharmacological activities, chemical constituents, and efficiencies in clinical application. To distinguish five main species of Rhodiola and ensure their safety and efficacy, microscopic characteristics of roots, rhizomes, and stems, including transverse sections, stem and foliar epidermis, as well as the crude drug powder, were observed. The fixed, sectioned, and stained plant materials, as well as the crude powder, were studied using a light microscope according to the usual microscopic techniques. The results of the microscopic features were systematically and comparatively described and illustrated. The five species have distinct microscopic characteristic differences, thus allowing us to distinguish between the species. Also, semi-quantitative and quantitative micrographic parameter tables were simultaneously presented. Further, a key to the five species and a comparative chart of the key authentication parameters based on these anatomic characteristics analyzed was drawn up and is presented for the Rhodiola species studied. The study indicated that light microscopy and related techniques provide a method that is convenient, feasible, and can be unambiguously applied to the authentication of species of Rhodiola. (c) 2008 Wiley-Liss, Inc.

Photoluminescence Imaging and LBIC Characterization of Defects in mc-Si Solar Cells

NASA Astrophysics Data System (ADS)

Sánchez, L. A.; Moretón, A.; Guada, M.; Rodríguez-Conde, S.; Martínez, O.; González, M. A.; Jiménez, J.

2018-05-01

Today's photovoltaic market is dominated by multicrystalline silicon (mc-Si) based solar cells with around 70% of worldwide production. In order to improve the quality of the Si material, a proper characterization of the electrical activity in mc-Si solar cells is essential. A full-wafer characterization technique such as photoluminescence imaging (PLi) provides a fast inspection of the wafer defects, though at the expense of the spatial resolution. On the other hand, a study of the defects at a microscopic scale can be achieved through the light-beam induced current technique. The combination of these macroscopic and microscopic resolution techniques allows a detailed study of the electrical activity of defects in mc-Si solar cells. In this work, upgraded metallurgical-grade Si solar cells are studied using these two techniques.

Thin laser light sheet microscope for microbial oceanography

NASA Astrophysics Data System (ADS)

Fuchs, Eran; Jaffe, Jules S.; Long, Richard A.; Azam, Farooq

2002-01-01

Despite a growing need, oceanographers are limited by existing technological constrains and are unable to observe aquatic microbes in their natural setting. In order to provide a simple and easy to implement solution for such studies, a new Thin Light Sheet Microscope (TLSM) has been developed. The TLSM utilizes a well-defined sheet of laser light, which has a narrow (23 micron) axial dimension over a 1 mm x 1 mm field of view. This light sheet is positioned precisely within the depth of field of the microscope’s objective lens. The technique thus utilizes conventional microscope optics but replaces the illumination system. The advantages of the TLSM are two-fold: First, it concentrates light only where excitation is needed, thus maximizing the efficiency of the illumination source. Secondly, the TLSM maximizes image sharpness while at the same time minimizing the level of background noise. Particles that are not located within the objective's depth of field are not illuminated and therefore do not contribute to an out-of-focus image. Images from a prototype system that used SYBR Green I fluorescence stain in order to localize single bacteria are reported. The bacteria were in a relatively large and undisturbed volume of 4ml, which contained natural seawater. The TLSM can be used for fresh water studies of bacteria with no modification. The microscope permits the observation of interactions at the microscale and has potential to yield insights into how microbes structure pelagic ecosystems.

Electron tomography of whole cultured cells using novel transmission electron imaging technique.

PubMed

Okumura, Taiga; Shoji, Minami; Hisada, Akiko; Ominami, Yusuke; Ito, Sukehiro; Ushiki, Tatsuo; Nakajima, Masato; Ohshima, Takashi

2018-01-01

Since a three-dimensional (3D) cellular ultrastructure is significant for biological functions, it has been investigated using various electron microscopic techniques. Although transmission electron microscopy (TEM)-based techniques are traditionally used, cells must be embedded in resin and sliced into ultrathin sections in sample preparation processes. Block-face observation using a scanning electron microscope (SEM) has also been recently applied to 3D observation of cellular components, but this is a destructive inspection and does not allow re-examination. Therefore, we developed electron tomography using a transmission electron imaging technique called Plate-TEM. With Plate-TEM, the cells cultured directly on a scintillator plate are inserted into a conventional SEM equipped with a Plate-TEM observation system, and their internal structures are observed by detecting scintillation light produced by electrons passing through the cells. This technology has the following four advantages. First, the cells cultured on the plate can be observed at electron-microscopic resolution since they remain on the plate. Second, both surface and internal information can be obtained simultaneously by using electron- and photo-detectors, respectively, because a Plate-TEM detector is installed in an SEM. Third, the cells on the scintillator plate can also be inspected using light microscopy because the plate has transparent features. Finally, correlative observation with other techniques, such as conventional TEM, is possible after Plate-TEM observation because Plate-TEM is a non-destructive analysis technique. We also designed a sample stage to tilt the samples for tomography with Plate-TEM, by which 3D organization of cellular structures can be visualized as a whole cell. In the present study, Mm2T cells were investigated using our tomography system, resulting in 3D visualization of cell organelles such as mitochondria, lipid droplets, and microvilli. Correlative observations with various imaging techniques were also conducted by successive observations with light microscopy, SEM, Plate-TEM, and conventional TEM. Consequently, the Plate-TEM tomography technique encourages understanding of cellular structures at high resolution, which can contribute to cellular biological research. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mathematical model of a DIC position sensing system within an optical trap

NASA Astrophysics Data System (ADS)

Wulff, Kurt D.; Cole, Daniel G.; Clark, Robert L.

2005-08-01

The quantitative study of displacements and forces of motor proteins and processes that occur at the microscopic level and below require a high level of sensitivity. For optical traps, two techniques for position sensing have been accepted and used quite extensively: quadrant photodiodes and an interferometric position sensing technique based on DIC imaging. While quadrant photodiodes have been studied in depth and mathematically characterized, a mathematical characterization of the interferometric position sensor has not been presented to the authors' knowledge. The interferometric position sensing method works off of the DIC imaging capabilities of a microscope. Circularly polarized light is sent into the microscope and the Wollaston prism used for DIC imaging splits the beam into its orthogonal components, displacing them by a set distance determined by the user. The distance between the axes of the beams is set so the beams overlap at the specimen plane and effectively share the trapped microsphere. A second prism then recombines the light beams and the exiting laser light's polarization is measured and related to position. In this paper we outline the mathematical characterization of a microsphere suspended in an optical trap using a DIC position sensing method. The sensitivity of this mathematical model is then compared to the QPD model. The mathematical model of a microsphere in an optical trap can serve as a calibration curve for an experimental setup.

SPIM-fluid: open source light-sheet based platform for high-throughput imaging

PubMed Central

Gualda, Emilio J.; Pereira, Hugo; Vale, Tiago; Estrada, Marta Falcão; Brito, Catarina; Moreno, Nuno

2015-01-01

Light sheet fluorescence microscopy has recently emerged as the technique of choice for obtaining high quality 3D images of whole organisms/embryos with low photodamage and fast acquisition rates. Here we present an open source unified implementation based on Arduino and Micromanager, which is capable of operating Light Sheet Microscopes for automatized 3D high-throughput imaging on three-dimensional cell cultures and model organisms like zebrafish, oriented to massive drug screening. PMID:26601007

Scanning electron microscope cathodoluminescence imaging of subgrain boundaries, twins and planar deformation features in quartz

NASA Astrophysics Data System (ADS)

Hamers, M. F.; Pennock, G. M.; Drury, M. R.

2017-04-01

The study of deformation features has been of great importance to determine deformation mechanisms in quartz. Relevant microstructures in both growth and deformation processes include dislocations, subgrains, subgrain boundaries, Brazil and Dauphiné twins and planar deformation features (PDFs). Dislocations and twin boundaries are most commonly imaged using a transmission electron microscope (TEM), because these cannot directly be observed using light microscopy, in contrast to PDFs. Here, we show that red-filtered cathodoluminescence imaging in a scanning electron microscope (SEM) is a useful method to visualise subgrain boundaries, Brazil and Dauphiné twin boundaries. Because standard petrographic thin sections can be studied in the SEM, the observed structures can be directly and easily correlated to light microscopy studies. In contrast to TEM preparation methods, SEM techniques are non-destructive to the area of interest on a petrographic thin section.

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection.

PubMed

Mertz, Jerome; Kim, Jinhyun

2010-01-01

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast.

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection

NASA Astrophysics Data System (ADS)

Mertz, Jerome; Kim, Jinhyun

2010-01-01

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast.

Generation of multiple Bessel beams for a biophotonics workstation.

PubMed

Cizmár, T; Kollárová, V; Tsampoula, X; Gunn-Moore, F; Sibbett, W; Bouchal, Z; Dholakia, K

2008-09-01

We present a simple method using an axicon and spatial light modulator to create multiple parallel Bessel beams and precisely control their individual positions in three dimensions. This technique is tested as an alternative to classical holographic beam shaping commonly used now in optical tweezers. Various applications of precise control of multiple Bessel beams are demonstrated within a single microscope giving rise to new methods for three-dimensional positional control of trapped particles or active sorting of micro-objects as well as "focus-free" photoporation of living cells. Overall this concept is termed a 'biophotonics workstation' where users may readily trap, sort and porate material using Bessel light modes in a microscope.

Light and Life in Baltimore—and Beyond

PubMed Central

Edidin, Michael

2015-01-01

Baltimore has been the home of numerous biophysical studies using light to probe cells. One such study, quantitative measurement of lateral diffusion of rhodopsin, set the standard for experiments in which recovery after photobleaching is used to measure lateral diffusion. Development of this method from specialized microscopes to commercial scanning confocal microscopes has led to widespread use of the technique to measure lateral diffusion of membrane proteins and lipids, and as well diffusion and binding interactions in cell organelles and cytoplasm. Perturbation of equilibrium distributions by photobleaching has also been developed into a robust method to image molecular proximity in terms of fluorescence resonance energy transfer between donor and acceptor fluorophores. PMID:25650914

Correlative light and electron microscopic detection of GFP-labeled proteins using modular APEX.

PubMed

Ariotti, Nicholas; Hall, Thomas E; Parton, Robert G

2017-01-01

The use of green fluorescent protein (GFP) and related proteins has revolutionized light microscopy. Here we describe a rapid and simple method to localize GFP-tagged proteins in cells and in tissues by electron microscopy (EM) using a modular approach involving a small GFP-binding peptide (GBP) fused to the ascorbate peroxidase-derived APEX2 tag. We provide a method for visualizing GFP-tagged proteins by light and EM in cultured cells and in the zebrafish using modular APEX-GBP. Furthermore, we describe in detail the benefits of this technique over many of the currently available correlative light and electron microscopy approaches and demonstrate APEX-GBP is readily applicable to modern three-dimensional techniques. Copyright © 2017 Elsevier Inc. All rights reserved.

Scanning thin-sheet laser imaging microscopy (sTSLIM) with structured illumination and HiLo background rejection.

PubMed

Schröter, Tobias J; Johnson, Shane B; John, Kerstin; Santi, Peter A

2012-01-01

We report replacement of one side of a static illumination, dual sided, thin-sheet laser imaging microscope (TSLIM) with an intensity modulated laser scanner in order to implement structured illumination (SI) and HiLo image demodulation techniques for background rejection. The new system is equipped with one static and one scanned light-sheet and is called a scanning thin-sheet laser imaging microscope (sTSLIM). It is an optimized version of a light-sheet fluorescent microscope that is designed to image large specimens (<15 mm in diameter). In this paper we describe the hardware and software modifications to TSLIM that allow for static and uniform light-sheet illumination with SI and HiLo image demodulation. The static light-sheet has a thickness of 3.2 µm; whereas, the scanned side has a light-sheet thickness of 4.2 µm. The scanned side images specimens with subcellular resolution (<1 µm lateral and <4 µm axial resolution) with a size up to 15 mm. SI and HiLo produce superior contrast compared to both the uniform static and scanned light-sheets. HiLo contrast was greater than SI and is faster and more robust than SI because as it produces images in two-thirds of the time and exhibits fewer intensity streaking artifacts. 2011 Optical Society of America

Parallel-multiplexed excitation light-sheet microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Xu, Dongli; Zhou, Weibin; Peng, Leilei

2017-02-01

Laser scanning light-sheet imaging allows fast 3D image of live samples with minimal bleach and photo-toxicity. Existing light-sheet techniques have very limited capability in multi-label imaging. Hyper-spectral imaging is needed to unmix commonly used fluorescent proteins with large spectral overlaps. However, the challenge is how to perform hyper-spectral imaging without sacrificing the image speed, so that dynamic and complex events can be captured live. We report wavelength-encoded structured illumination light sheet imaging (λ-SIM light-sheet), a novel light-sheet technique that is capable of parallel multiplexing in multiple excitation-emission spectral channels. λ-SIM light-sheet captures images of all possible excitation-emission channels in true parallel. It does not require compromising the imaging speed and is capable of distinguish labels by both excitation and emission spectral properties, which facilitates unmixing fluorescent labels with overlapping spectral peaks and will allow more labels being used together. We build a hyper-spectral light-sheet microscope that combined λ-SIM with an extended field of view through Bessel beam illumination. The system has a 250-micron-wide field of view and confocal level resolution. The microscope, equipped with multiple laser lines and an unlimited number of spectral channels, can potentially image up to 6 commonly used fluorescent proteins from blue to red. Results from in vivo imaging of live zebrafish embryos expressing various genetic markers and sensors will be shown. Hyper-spectral images from λ-SIM light-sheet will allow multiplexed and dynamic functional imaging in live tissue and animals.

Introduction to Modern Methods in Light Microscopy.

PubMed

Ryan, Joel; Gerhold, Abby R; Boudreau, Vincent; Smith, Lydia; Maddox, Paul S

2017-01-01

For centuries, light microscopy has been a key method in biological research, from the early work of Robert Hooke describing biological organisms as cells, to the latest in live-cell and single-molecule systems. Here, we introduce some of the key concepts related to the development and implementation of modern microscopy techniques. We briefly discuss the basics of optics in the microscope, super-resolution imaging, quantitative image analysis, live-cell imaging, and provide an outlook on active research areas pertaining to light microscopy.

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.

Label-free tracking of single extracellular vesicles in a nano-fluidic optical fiber (Conference Presentation)

NASA Astrophysics Data System (ADS)

van der Pol, Edwin; Weidlich, Stefan; Lahini, Yoav; Coumans, Frank A. W.; Sturk, Auguste; Nieuwland, Rienk; Schmidt, Markus A.; Faez, Sanli; van Leeuwen, Ton G.

2016-03-01

Background: Extracellular vesicles, such as exosomes, are abundantly present in human body fluids. Since the size, concentration and composition of these vesicles change during disease, vesicles have promising clinical applications, including cancer diagnosis. However, since ~70% of the vesicles have a diameter <70 nm, detection of single vesicles remains challenging. Thus far, vesicles <70 nm have only be studied by techniques that require the vesicles to be adhered to a surface. Consequently, the majority of vesicles have never been studied in their physiological environment. We present a novel label-free optical technique to track single vesicles <70 nm in suspension. Method: Urinary vesicles were contained within a single-mode light-guiding silica fiber containing a 600 nm nano-fluidic channel. Light from a diode laser (660 nm wavelength) was coupled to the fiber, resulting in a strongly confined optical mode in the nano-fluidic channel, which continuously illuminated the freely diffusing vesicles inside the channel. The elastic light scattering from the vesicles, in the direction orthogonal to the fiber axis, was collected using a microscope objective (NA=0.95) and imaged with a home-built microscope. Results: We have tracked single urinary vesicles as small as 35 nm by elastic light scattering. Please note that vesicles are low-refractive index (n<1.4) particles, which we confirmed by combining data on thermal diffusion and light scattering cross section. Conclusions: For the first time, we have studied vesicles <70 nm freely diffusing in suspension. The ease-of-use and performance of this technique support its potential for vesicle-based clinical applications.

Confocal Fluorescence Microscopy of Mung Beanleaves

NASA Astrophysics Data System (ADS)

Chen, Zhiwei; Liu, Dongwu

Recently, confocal microscope has become a routine technique and indispensable tool for cell biological studies and molecular investigations. The light emitted from the point out-of-focus is blocked by the pinhole and can not reach the detector, which is one of the critical features of the confocal microscope. In present studies, the probes acridine orange (AO) and rhodamine-123 were used to research stoma and mitochondria of mung bean leaves, respectively. The results indicated that the stomatal guard cells and mitochondria were clearly seen in epidermic tissue of mung bean leaves. Taken together, it is a good method to research plant cells with confocal microscope and fluorescence probes.

Infrared absorption nano-spectroscopy using sample photoexpansion induced by tunable quantum cascade lasers.

PubMed

Lu, Feng; Belkin, Mikhail A

2011-10-10

We report a simple technique that allows obtaining mid-infrared absorption spectra with nanoscale spatial resolution under low-power illumination from tunable quantum cascade lasers. Light absorption is detected by measuring associated sample thermal expansion with an atomic force microscope. To detect minute thermal expansion we tune the repetition frequency of laser pulses in resonance with the mechanical frequency of the atomic force microscope cantilever. Spatial resolution of better than 50 nm is experimentally demonstrated.

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

PubMed

Paddock, Stephen W; Eliceiri, Kevin W

2014-01-01

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

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection

PubMed Central

Mertz, Jerome; Kim, Jinhyun

2010-01-01

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast. PMID:20210471

Diffraction Pattern Analysis as an Optical Inspection Technique

DTIC Science & Technology

1991-08-01

BACKGROUND Diameters of fiber samples have commonly been measured manually with an optical microscope. Marcuse and Presby developed an automatic...by analyzing the back-scattered light when a beam of laser light impinged upon the fiber [2]. Presby and Marcuse extended this back-scattering tech...be im- proved further in order to become a feasible method for detecting a small number of blocked openings in CRT screens. 20 REFERENCES 1. Marcuse

Novel approach to real-time flash photolysis and confocal [Ca2+] imaging

PubMed Central

Sobie, Eric A.; Kao, Joseph P.Y.; Lederer, W. J.

2008-01-01

Flash photolysis of “caged” compounds using ultraviolet light is a powerful experimental technique for producing rapid changes in concentrations of bioactive signaling molecules. Studies that employ this technique have used diverse strategies for controlling the spatial and temporal application of light to the specimen. Here we describe a new system for flash photolysis that delivers light from a pulsed, adjustable intensity laser through an optical fiber coupled into the epifluorescence port of a commercial confocal microscope. Photolysis is achieved with extremely brief (5 ns) pulses of ultraviolet light (355 nm) that can be synchronized with respect to confocal laser scanning. The system described also localizes the UV intensity spatially so that uncaging only occurs in defined sub-cellular regions; moreover, since the microscope optics are used in localization, the photolysis volume can be easily adjusted. Experiments performed on rat ventricular myocytes loaded with the Ca2+ indicator fluo-3 and the Ca2+ cage NP-EGTA demonstrate the system's capabilities. Localized intracellular increases in [Ca2+] can trigger sarcoplasmic reticular Ca2+ release events such as Ca2+ sparks and, under certain conditions, regenerative Ca2+ waves. This relatively simple and inexpensive system is therefore a useful tool for examining local signaling in heart and other tissues. PMID:17323075

A simple backscattering microscope for fast tracking of biological molecules

PubMed Central

Sowa, Yoshiyuki; Steel, Bradley C.; Berry, Richard M.

2010-01-01

Recent developments in techniques for observing single molecules under light microscopes have helped reveal the mechanisms by which molecular machines work. A wide range of markers can be used to detect molecules, from single fluorophores to micron sized markers, depending on the research interest. Here, we present a new and simple objective-type backscattering microscope to track gold nanoparticles with nanometer and microsecond resolution. The total noise of our system in a 55 kHz bandwidth is ∼0.6 nm per axis, sufficient to measure molecular movement. We found our backscattering microscopy to be useful not only for in vitro but also for in vivo experiments because of lower background scattering from cells than in conventional dark-field microscopy. We demonstrate the application of this technique to measuring the motion of a biological rotary molecular motor, the bacterial flagellar motor, in live Escherichia coli cells. PMID:21133475

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.

Light and life in Baltimore--and beyond.

PubMed

Edidin, Michael

2015-02-03

Baltimore has been the home of numerous biophysical studies using light to probe cells. One such study, quantitative measurement of lateral diffusion of rhodopsin, set the standard for experiments in which recovery after photobleaching is used to measure lateral diffusion. Development of this method from specialized microscopes to commercial scanning confocal microscopes has led to widespread use of the technique to measure lateral diffusion of membrane proteins and lipids, and as well diffusion and binding interactions in cell organelles and cytoplasm. Perturbation of equilibrium distributions by photobleaching has also been developed into a robust method to image molecular proximity in terms of fluorescence resonance energy transfer between donor and acceptor fluorophores. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Stress Measurement by Geometrical Optics

NASA Technical Reports Server (NTRS)

Robinson, R. S.; Rossnagel, S. M.

1986-01-01

Fast, simple technique measures stresses in thin films. Sample disk bowed by stress into approximately spherical shape. Reflected image of disk magnified by amount related to curvature and, therefore, stress. Method requires sample substrate, such as cheap microscope cover slide, two mirrors, laser light beam, and screen.

Low-cost cryo-light microscopy stage fabrication for correlated light/electron microscopy.

PubMed

Carlson, David B; Evans, James E

2011-06-05

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

The influence of the microscope lamp filament colour temperature on the process of digital images of histological slides acquisition standardization.

PubMed

Korzynska, Anna; Roszkowiak, Lukasz; Pijanowska, Dorota; Kozlowski, Wojciech; Markiewicz, Tomasz

2014-01-01

The aim of this study is to compare the digital images of the tissue biopsy captured with optical microscope using bright field technique under various light conditions. The range of colour's variation in immunohistochemically stained with 3,3'-Diaminobenzidine and Haematoxylin tissue samples is immense and coming from various sources. One of them is inadequate setting of camera's white balance to microscope's light colour temperature. Although this type of error can be easily handled during the stage of image acquisition, it can be eliminated with use of colour adjustment algorithms. The examination of the dependence of colour variation from microscope's light temperature and settings of the camera is done as an introductory research to the process of automatic colour standardization. Six fields of view with empty space among the tissue samples have been selected for analysis. Each field of view has been acquired 225 times with various microscope light temperature and camera white balance settings. The fourteen randomly chosen images have been corrected and compared, with the reference image, by the following methods: Mean Square Error, Structural SIMilarity and visual assessment of viewer. For two types of backgrounds and two types of objects, the statistical image descriptors: range, median, mean and its standard deviation of chromaticity on a and b channels from CIELab colour space, and luminance L, and local colour variability for objects' specific area have been calculated. The results have been averaged for 6 images acquired in the same light conditions and camera settings for each sample. The analysis of the results leads to the following conclusions: (1) the images collected with white balance setting adjusted to light colour temperature clusters in certain area of chromatic space, (2) the process of white balance correction for images collected with white balance camera settings not matched to the light temperature moves image descriptors into proper chromatic space but simultaneously the value of luminance changes. So the process of the image unification in a sense of colour fidelity can be solved in separate introductory stage before the automatic image analysis.

Coherent beam control through inhomogeneous media in multi-photon microscopy

NASA Astrophysics Data System (ADS)

Paudel, Hari Prasad

Multi-photon fluorescence microscopy has become a primary tool for high-resolution deep tissue imaging because of its sensitivity to ballistic excitation photons in comparison to scattered excitation photons. The imaging depth of multi-photon microscopes in tissue imaging is limited primarily by background fluorescence that is generated by scattered light due to the random fluctuations in refractive index inside the media, and by reduced intensity in the ballistic focal volume due to aberrations within the tissue and at its interface. We built two multi-photon adaptive optics (AO) correction systems, one for combating scattering and aberration problems, and another for compensating interface aberrations. For scattering correction a MEMS segmented deformable mirror (SDM) was inserted at a plane conjugate to the objective back-pupil plane. The SDM can pre-compensate for light scattering by coherent combination of the scattered light to make an apparent focus even at a depths where negligible ballistic light remains (i.e. ballistic limit). This problem was approached by investigating the spatial and temporal focusing characteristics of a broad-band light source through strongly scattering media. A new model was developed for coherent focus enhancement through or inside the strongly media based on the initial speckle contrast. A layer of fluorescent beads under a mouse skull was imaged using an iterative coherent beam control method in the prototype two-photon microscope to demonstrate the technique. We also adapted an AO correction system to an existing in three-photon microscope in a collaborator lab at Cornell University. In the second AO correction approach a continuous deformable mirror (CDM) is placed at a plane conjugate to the plane of an interface aberration. We demonstrated that this "Conjugate AO" technique yields a large field-of-view (FOV) advantage in comparison to Pupil AO. Further, we showed that the extended FOV in conjugate AO is maintained over a relatively large axial misalignment of the conjugate planes of the CDM and the aberrating interface. This dissertation advances the field of microscopy by providing new models and techniques for imaging deeply within strongly scattering tissue, and by describing new adaptive optics approaches to extending imaging FOV due to sample aberrations.

The role of light microscopy in aerospace analytical laboratories

NASA Technical Reports Server (NTRS)

Crutcher, E. R.

1977-01-01

Light microscopy has greatly reduced analytical flow time and added new dimensions to laboratory capability. Aerospace analytical laboratories are often confronted with problems involving contamination, wear, or material inhomogeneity. The detection of potential problems and the solution of those that develop necessitate the most sensitive and selective applications of sophisticated analytical techniques and instrumentation. This inevitably involves light microscopy. The microscope can characterize and often identify the cause of a problem in 5-15 minutes with confirmatory tests generally less than one hour. Light microscopy has and will make a very significant contribution to the analytical capabilities of aerospace laboratories.

All-photonic drying and sintering process via flash white light combined with deep-UV and near-infrared irradiation for highly conductive copper nano-ink

PubMed Central

Hwang, Hyun-Jun; Oh, Kyung-Hwan; Kim, Hak-Sung

2016-01-01

We developed an ultra-high speed photonic sintering method involving flash white light (FWL) combined with near infrared (NIR) and deep UV light irradiation to produce highly conductive copper nano-ink film. Flash white light irradiation energy and the power of NIR/deep UV were optimized to obtain high conductivity Cu films. Several microscopic and spectroscopic characterization techniques such as scanning electron microscopy (SEM), a x-ray diffraction (XRD), and Fourier-transform infrared (FT-IR) spectroscopy were employed to characterize the Cu nano-films. Optimally sintered Cu nano-ink films produced using a deep UV-assisted flash white light sintering technique had the lowest resistivity (7.62 μΩ·cm), which was only 4.5-fold higher than that of bulk Cu film (1.68 μΩ•cm). PMID:26806215

All-photonic drying and sintering process via flash white light combined with deep-UV and near-infrared irradiation for highly conductive copper nano-ink.

PubMed

Hwang, Hyun-Jun; Oh, Kyung-Hwan; Kim, Hak-Sung

2016-01-25

We developed an ultra-high speed photonic sintering method involving flash white light (FWL) combined with near infrared (NIR) and deep UV light irradiation to produce highly conductive copper nano-ink film. Flash white light irradiation energy and the power of NIR/deep UV were optimized to obtain high conductivity Cu films. Several microscopic and spectroscopic characterization techniques such as scanning electron microscopy (SEM), a x-ray diffraction (XRD), and Fourier-transform infrared (FT-IR) spectroscopy were employed to characterize the Cu nano-films. Optimally sintered Cu nano-ink films produced using a deep UV-assisted flash white light sintering technique had the lowest resistivity (7.62 μΩ·cm), which was only 4.5-fold higher than that of bulk Cu film (1.68 μΩ•cm).

Optical improvements in the diagnosis of bladder cancer: implications for clinical practice.

PubMed

Schubert, Tina; Rausch, Steffen; Fahmy, Omar; Gakis, Georgios; Stenzl, Arnulf

2017-11-01

For over 100 years white-light cystoscopy has remained the gold-standard technique for the detection of bladder cancer (BCa). Some limitations in the detection of flat lesions (CIS), the differentiation between inflammation and malignancy, the inaccurate determination of the tumor margin status as well as the tumor depth, have led to a variety of technological improvements. The aim of this review is to evaluate the impact of these improvements in the diagnosis of BCa and their effectiveness in clinical practice. A systematic literature search was conducted according to the PRISMA statement to identify studies reporting on imaging modalities in the diagnosis of NMIBC between 2000 and 2017. A two-stage selection process was utilized to determine eligible studies. A total of 74 studies were considered for final analysis. Optical imaging technologies have emerged as an adjunct to white-light cystoscopy and can be classified according to their scope as macroscopic, microscopic and molecular. Macroscopic techniques including photodynamic diagnosis (PDD), narrow-band imaging (NBI) and the Storz Professional Image Enhancement System (IMAGE1 S, formerly known as SPIES) are similar to white-light cystoscopy, but are superior in the detection of bladder tumors by means of contrast enhancement. Especially the detection rate of very mute lesions in the bladder mucosa (CIS) could be significantly increased by the use of these methods. Microscopic imaging techniques like confocal laser endomicroscopy and optical coherence tomography permit a real-time high-resolution assessment of the bladder mucosa at a cellular and sub-cellular level with spatial resolutions similar to histology, enabling the surgeon to perform an 'optical biopsy'. Molecular techniques are based on the combination of optical imaging technologies with fluorescence labeling of cancer-specific molecular agents like antibodies. This labeling is intended to favor an optical distinction between benign and malignant tissue. Optical improvements of the standard white-light cystoscopy have proven their benefit in the detection of BCa and have found their way into clinical practice. Especially the combination of macroscopic and microscopic techniques may improve diagnostic accuracy. Nevertheless, HAL-PDD guided cystoscopy is the only approach approved for routine use in the diagnosis of BCa by most urological associations in the EU and USA to date.

Ophthalmic applications of confocal microscopy: diagnostics, refractive surgery, and eye banking

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1990-11-01

Confocal microscopy of ocular tissue provides two advantages over traditional imaging techniques: increased range and transverse resolution and increased contrast. The semitransparent cornea and ocular lens in the living eye can be optically sectioned and observed by reflected light confocal microscopy. Within the cornea we observed various cell components nerve fibers nerve cell bodies and fibrous networks. The confocal microscopic images from the in-situ ocular lens show the lens capsule the lens epithelium and the individual lens fibrils. All of the reflected light confocal microscopic images have high contrast and high resolution. Some of the applications of confocal imaging in ophthalmology include: diagnostics of the cornea and the ocular lens examination prior to and after refractive surgery examination of intraocular lenses (IOL) and examination of eye bank material. Other ophthalmic uses of confocal imaging include: studies of wound healing therapeutics and the effects of contact lenses on the cornea. The proposed features of a clinical confocal microscope are reviewed. 2.

Photonic Microhand with Autonomous Action.

PubMed

Martella, Daniele; Nocentini, Sara; Nuzhdin, Dmitry; Parmeggiani, Camilla; Wiersma, Diederik S

2017-11-01

Grabbing and holding objects at the microscale is a complex function, even for microscopic living animals. Inspired by the hominid-type hand, a microscopic equivalent able to catch microelements is engineered. This microhand is light sensitive and can be either remotely controlled by optical illumination or can act autonomously and grab small particles on the basis of their optical properties. Since the energy is delivered optically, without the need for wires or batteries, the artificial hand can be shrunk down to the micrometer scale. Soft material is used, in particular, a custom-made liquid-crystal network that is patterned by a photolithographic technique. The elastic reshaping properties of this material allow finger movement, using environmental light as the only energy source. The hand can be either controlled externally (via the light field), or else the conditions in which it autonomously grabs a particle in its vicinity can be created. This microrobot has the unique feature that it can distinguish between particles of different colors and gray levels. The realization of this autonomous hand constitutes a crucial element in the development of microscopic creatures that can perform tasks without human intervention and self-organized automation at the micrometer scale. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Novel Optical Technique Developed and Tested for Measuring Two-Point Velocity Correlations in Turbulent Flows

NASA Technical Reports Server (NTRS)

Zimmerli, Gregory A.; Goldburg, Walter I.

2002-01-01

A novel technique for characterizing turbulent flows was developed and tested at the NASA Glenn Research Center. The work is being done in collaboration with the University of Pittsburgh, through a grant from the NASA Microgravity Fluid Physics Program. The technique we are using, Homodyne Correlation Spectroscopy (HCS), is a laser-light-scattering technique that measures the Doppler frequency shift of light scattered from microscopic particles in the fluid flow. Whereas Laser Doppler Velocimetry gives a local (single-point) measurement of the fluid velocity, the HCS technique measures correlations between fluid velocities at two separate points in the flow at the same instant of time. Velocity correlations in the flow field are of fundamental interest to turbulence researchers and are of practical importance in many engineering applications, such as aeronautics.

Spectrally resolved laser interference microscopy

NASA Astrophysics Data System (ADS)

Butola, Ankit; Ahmad, Azeem; Dubey, Vishesh; Senthilkumaran, P.; Singh Mehta, Dalip

2018-07-01

We developed a new quantitative phase microscopy technique, namely, spectrally resolved laser interference microscopy (SR-LIM), with which it is possible to quantify multi-spectral phase information related to biological specimens without color crosstalk using a color CCD camera. It is a single shot technique where sequential switched on/off of red, green, and blue (RGB) wavelength light sources are not required. The method is implemented using a three-wavelength interference microscope and a customized compact grating based imaging spectrometer fitted at the output port. The results of the USAF resolution chart while employing three different light sources, namely, a halogen lamp, light emitting diodes, and lasers, are discussed and compared. The broadband light sources like the halogen lamp and light emitting diodes lead to stretching in the spectrally decomposed images, whereas it is not observed in the case of narrow-band light sources, i.e. lasers. The proposed technique is further successfully employed for single-shot quantitative phase imaging of human red blood cells at three wavelengths simultaneously without color crosstalk. Using the present technique, one can also use a monochrome camera, even though the experiments are performed using multi-color light sources. Finally, SR-LIM is not only limited to RGB wavelengths, it can be further extended to red, near infra-red, and infra-red wavelengths, which are suitable for various biological applications.

Integration of a high-NA light microscope in a scanning electron microscope.

PubMed

Zonnevylle, A C; Van Tol, R F C; Liv, N; Narvaez, A C; Effting, A P J; Kruit, P; Hoogenboom, J P

2013-10-01

We present an integrated light-electron microscope in which an inverted high-NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high-resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub-10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum-compatible immersion oil. For a 40-nm-diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

Laser based imaging of time depending microscopic scenes with strong light emission

NASA Astrophysics Data System (ADS)

Hahlweg, Cornelius; Wilhelm, Eugen; Rothe, Hendrik

2011-10-01

Investigating volume scatterometry methods based on short range LIDAR devices for non-static objects we achieved interesting results aside the intended micro-LIDAR: the high speed camera recording of the illuminated scene of an exploding wire -intended for Doppler LIDAR tests - delivered a very effective method of observing details of objects with extremely strong light emission. As a side effect a schlieren movie is gathered without any special effort. The fact that microscopic features of short time processes with high emission and material flow might be imaged without endangering valuable equipment makes this technique at least as interesting as the intended one. So we decided to present our results - including latest video and photo material - instead of a more theoretical paper on our progress concerning the primary goal.

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.

eduSPIM: Light Sheet Microscopy in the Museum.

PubMed

Jahr, Wiebke; Schmid, Benjamin; Weber, Michael; Huisken, Jan

2016-01-01

Light sheet microscopy (or selective plane illumination microscopy) is an important imaging technique in the life sciences. At the same time, this technique is also ideally suited for community outreach projects, because it produces visually appealing, highly dynamic images of living organisms and its working principle can be understood with basic optics knowledge. Still, the underlying concepts are widely unknown to the non-scientific public. On the occasion of the UNESCO International Year of Light, a technical museum in Dresden, Germany, launched a special, interactive exhibition. We built a fully functional, educational selective plane illumination microscope (eduSPIM) to demonstrate how developments in microscopy promote discoveries in biology. To maximize educational impact, we radically reduced a standard light sheet microscope to its essential components without compromising functionality and incorporated stringent safety concepts beyond those needed in the lab. Our eduSPIM system features one illumination and one detection path and a sealed sample chamber. We image fixed zebrafish embryos with fluorescent vasculature, because the structure is meaningful to laymen and visualises the optical principles of light sheet microscopy. Via a simplified interface, visitors acquire fluorescence and transmission data simultaneously. The universal concepts presented here may also apply to other scientific approaches that are communicated to laymen in interactive settings. The specific eduSPIM design is adapted easily for various outreach and teaching activities. eduSPIM may even prove useful for labs needing a simple SPIM. A detailed parts list and schematics to rebuild eduSPIM are provided.

Staining-free malaria diagnostics by multispectral and multimodality light-emitting-diode microscopy

NASA Astrophysics Data System (ADS)

Merdasa, Aboma; Brydegaard, Mikkel; Svanberg, Sune; Zoueu, Jeremie T.

2013-03-01

We report an accurate optical differentiation technique between healthy and malaria-infected erythrocytes by quasi-simultaneous measurements of transmittance, reflectance, and scattering properties of unstained blood smears using a multispectral and multimode light-emitting diode microscope. We propose a technique for automated imaging, identification, and counting of malaria-infected erythrocytes for real-time and cost-effective parasitaemia diagnosis as an effective alternative to the manual screening of stained blood smears, now considered to be the gold standard in malaria diagnosis. We evaluate the performance of our algorithm against manual estimations of an expert and show a spectrally resolved increased scattering from malaria-infected blood cells.

Optical depth localization of nitrogen-vacancy centers in diamond with nanometer accuracy.

PubMed

Häußler, Andreas J; Heller, Pascal; McGuinness, Liam P; Naydenov, Boris; Jelezko, Fedor

2014-12-01

Precise positioning of nitrogen-vacancy (NV) centers is crucial for their application in sensing and quantum information. Here we present a new purely optical technique enabling determination of the NV position with nanometer resolution. We use a confocal microscope to determine the position of individual emitters along the optical axis. Using two separate detection channels, it is possible to simultaneously measure reflected light from the diamond surface and fluorescent light from the NV center and statistically evaluate both signals. An accuracy of 2.6 nm for shallow NV centers was achieved and is consistent with other techniques for depth determination.

Effect of operating microscope light on brain temperature during craniotomy.

PubMed

Gayatri, Parthasarathi; Menon, Girish G; Suneel, Puthuvassery R

2013-07-01

Operating microscopes used during neurosurgery are fitted with xenon light. Burn injuries have been reported because of xenon microscope lighting as the intensity of xenon light is 300 W. We designed this study to find out if the light of operating microscope causes an increase in temperature of the brain tissue, which is exposed underneath. Twenty-one adult patients scheduled for elective craniotomies were enrolled. Distal esophageal temperature (T Eso), brain temperature under the microscope light (T Brain), and brain temperature under dura mater (T Dura) were measured continuously at 15-minute intervals during microscope use. The irrigation fluid temperature, room temperature, intensity of the microscope light, and the distance of the microscope from the brain surface were kept constant. The average age of the patients was 44±15 years (18 males and 3 females). The mean duration of microscope use was 140±39 minutes. There were no significant changes in T Brain and T Dura and T Eso over time. T Dura was significantly lower than T Brain both at time 0 and 60 minutes but not at 90 minutes. T Brain was significantly lower than T Eso both at time 0 and 60 minutes but not at 90 minutes. The T Dura remained significantly lower than T Eso at 0, 60, and 90 minutes. Our study shows that there is no significant rise in brain temperature under xenon microscope light up to 120 minutes duration, at intensity of 60% to 70%, from a distance of 20 to 25 cm from the brain surface.

3D geometric phase analysis and its application in 3D microscopic morphology measurement

NASA Astrophysics Data System (ADS)

Zhu, Ronghua; Shi, Wenxiong; Cao, Quankun; Liu, Zhanwei; Guo, Baoqiao; Xie, Huimin

2018-04-01

Although three-dimensional (3D) morphology measurement has been widely applied on the macro-scale, there is still a lack of 3D measurement technology on the microscopic scale. In this paper, a microscopic 3D measurement technique based on the 3D-geometric phase analysis (GPA) method is proposed. In this method, with machine vision and phase matching, the traditional GPA method is extended to three dimensions. Using this method, 3D deformation measurement on the micro-scale can be realized using a light microscope. Simulation experiments were conducted in this study, and the results demonstrate that the proposed method has a good anti-noise ability. In addition, the 3D morphology of the necking zone in a tensile specimen was measured, and the results demonstrate that this method is feasible.

In vitro monitoring of oxidative processes with self-aggregating gold nanoparticles using all-optical photoacoustic spectroscopy.

PubMed

Yasmin, Zannatul; Khachatryan, Edward; Lee, Yuan-Hao; Maswadi, Saher; Glickman, Randolph; Nash, Kelly L

2015-02-15

In this work, the assembly of gold nanoparticles of (AuNPs) is used to detect the presence of the biomolecule glutathione (GSH) using a novel technique called "all-optical photoacoustic spectroscopy" (AOPAS). The AOPAS technique coupled with AuNPs forms the basis of a biosensing technique capable of probing the dynamic evolution of nano-bio interfaces within a microscopic volume. Dynamic Light Scattering (DLS) and ultraviolet-visible (UV-vis) spectra were measured to describe the kinetics governing the interparticle interactions by monitoring the AuNPs assembly and evolution of the surface plasmon resonance (SPR) band. A comparison of the same dynamic evolution of AuNPs assembly was performed using the AOPAS technique to confirm the validity of this method. The fundamental study is complemented by a demonstration of the performance of this biosensing technique in the presence of cell culture medium containing fetal bovine serum (FBS), which forms a protein corona on the surface of the AuNPs. This work demonstrates that the in vitro monitoring capabilities of the AOPAS provides sensitive measurement at the microscopic level and low nanoparticle concentrations without the artifacts limiting the use of conventional biosensing methods, such as fluorescent indicators. The AOPAS technique not only provides a facile approach for in vitro biosensing, but also shed a light on the real-time detection of thiol containing oxidative stress biomarkers in live systems using AuNPs. Copyright © 2014 Elsevier B.V. All rights reserved.

An historical account of the development and applications of the negative staining technique to the electron microscopy of viruses.

PubMed

Horne, R W; Wildy, P

1979-09-01

A brief historical account of the development and applications of the negative staining techniques to the study of the structure of viruses and their components as observed in the electron microscope is presented. Although the basic method of surrounding or embedding specimens in opaque dyes was used in light microscopy dating from about 1884, the equivalent preparative techniques applied to electron microscopy were comparatively recent. The combination of experiments on a sophisticated bacterial virus and the installation of a high resolution electron microscope in the Cavendish Laboratory, Cambridge, during 1954, subsequently led to the analysis of several important morphological features of animal, plant and bacterial viruses. The implications of the results from these early experiments on viruses and recent developments in negative staining methods for high resolution image analysis of electron micrographs are also discussed.

Optical method for high magnification imaging and video recording of live cells at sub-micron resolution

NASA Astrophysics Data System (ADS)

Romo, Jaime E., Jr.

Optical microscopy, the most common technique for viewing living microorganisms, is limited in resolution by Abbe's criterion. Recent microscopy techniques focus on circumnavigating the light diffraction limit by using different methods to obtain the topography of the sample. Systems like the AFM and SEM provide images with fields of view in the nanometer range with high resolvable detail, however these techniques are expensive, and limited in their ability to document live cells. The Dino-Lite digital microscope coupled with the Zeiss Axiovert 25 CFL microscope delivers a cost-effective method for recording live cells. Fields of view ranging from 8 microns to 300 microns with fair resolution provide a reliable method for discovering native cell structures at the nanoscale. In this report, cultured HeLa cells are recorded using different optical configurations resulting in documentation of cell dynamics at high magnification and resolution.

Quantitative readout of optically encoded gold nanorods using an ordinary dark-field microscope.

PubMed

Mercatelli, Raffaella; Ratto, Fulvio; Centi, Sonia; Soria, Silvia; Romano, Giovanni; Matteini, Paolo; Quercioli, Franco; Pini, Roberto; Fusi, Franco

2013-10-21

In this paper we report on a new use for dark-field microscopy in order to retrieve two-dimensional maps of optical parameters of a thin sample such as a cryptograph, a histological section, or a cell monolayer. In particular, we discuss the construction of quantitative charts of light absorbance and scattering coefficients of a polyvinyl alcohol film that was embedded with gold nanorods and then etched using a focused mode-locked Ti:Sapphire oscillator. Individual pulses from this laser excite plasmonic oscillations of the gold nanorods, thus triggering plastic deformations of the particles and their environment, which are confined within a few hundred nm of the light focus. In turn, these deformations modify the light absorbance and scattering landscape, which can be measured with optical resolution in a dark-field microscope equipped with an objective of tuneable numerical aperture. This technique may prove to be valuable for various applications, such as the fast readout of optically encoded data or to model functional interactions between light and biological tissue at the level of cellular organelles, including the photothermolysis of cancer.

Polarized Light Microscopy

NASA Technical Reports Server (NTRS)

Frandsen, Athela F.

2016-01-01

Polarized light microscopy (PLM) is a technique which employs the use of polarizing filters to obtain substantial optical property information about the material which is being observed. This information can be combined with other microscopy techniques to confirm or elucidate the identity of an unknown material, determine whether a particular contaminant is present (as with asbestos analysis), or to provide important information that can be used to refine a manufacturing or chemical process. PLM was the major microscopy technique in use for identification of materials for nearly a century since its introduction in 1834 by William Fox Talbot, as other techniques such as SEM (Scanning Electron Microscopy), FTIR (Fourier Transform Infrared spectroscopy), XPD (X-ray Powder Diffraction), and TEM (Transmission Electron Microscopy) had not yet been developed. Today, it is still the only technique approved by the Environmental Protection Agency (EPA) for asbestos analysis, and is often the technique first applied for identification of unknown materials. PLM uses different configurations in order to determine different material properties. With each configuration additional clues can be gathered, leading to a conclusion of material identity. With no polarizing filter, the microscope can be used just as a stereo optical microscope, and view qualities such as morphology, size, and number of phases. With a single polarizing filter (single polars), additional properties can be established, such as pleochroism, individual refractive indices, and dispersion staining. With two polarizing filters (crossed polars), even more can be deduced: isotropy vs. anisotropy, extinction angle, birefringence/degree of birefringence, sign of elongation, and anomalous polarization colors, among others. With the use of PLM many of these properties can be determined in a matter of seconds, even for those who are not highly trained. McCrone, a leader in the field of polarized light microscopy, often advised, If you cant determine a specific optical property of a particle after two minutes, move onto another configuration. Since optical properties can be seen so very quickly and easily under polarized light, it is only necessary to spend a maximum of two minutes on a technique to determine a particular property, though often only a few seconds are required.

Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy

PubMed Central

Wu, Yicong; Chandris, Panagiotis; Winter, Peter W.; Kim, Edward Y.; Jaumouillé, Valentin; Kumar, Abhishek; Guo, Min; Leung, Jacqueline M.; Smith, Corey; Rey-Suarez, Ivan; Liu, Huafeng; Waterman, Clare M.; Ramamurthi, Kumaran S.; La Riviere, Patrick J.; Shroff, Hari

2016-01-01

Most fluorescence microscopes are inefficient, collecting only a small fraction of the emitted light at any instant. Besides wasting valuable signal, this inefficiency also reduces spatial resolution and causes imaging volumes to exhibit significant resolution anisotropy. We describe microscopic and computational techniques that address these problems by simultaneously capturing and subsequently fusing and deconvolving multiple specimen views. Unlike previous methods that serially capture multiple views, our approach improves spatial resolution without introducing any additional illumination dose or compromising temporal resolution relative to conventional imaging. When applying our methods to single-view wide-field or dual-view light-sheet microscopy, we achieve a twofold improvement in volumetric resolution (~235 nm × 235 nm × 340 nm) as demonstrated on a variety of samples including microtubules in Toxoplasma gondii, SpoVM in sporulating Bacillus subtilis, and multiple protein distributions and organelles in eukaryotic cells. In every case, spatial resolution is improved with no drawback by harnessing previously unused fluorescence. PMID:27761486

Optical diffraction tomography with fully and partially coherent illumination in high numerical aperture label-free microscopy [Invited].

PubMed

Soto, Juan M; Rodrigo, José A; Alieva, Tatiana

2018-01-01

Quantitative label-free imaging is an important tool for the study of living microorganisms that, during the last decade, has attracted wide attention from the optical community. Optical diffraction tomography (ODT) is probably the most relevant technique for quantitative label-free 3D imaging applied in wide-field microscopy in the visible range. The ODT is usually performed using spatially coherent light illumination and specially designed holographic microscopes. Nevertheless, the ODT is also compatible with partially coherent illumination and can be realized in conventional wide-field microscopes by applying refocusing techniques, as it has been recently demonstrated. Here, we compare these two ODT modalities, underlining their pros and cons and discussing the optical setups for their implementation. In particular, we pay special attention to a system that is compatible with a conventional wide-field microscope that can be used for both ODT modalities. It consists of two easily attachable modules: the first for sample illumination engineering based on digital light processing technology; the other for focus scanning by using an electrically driven tunable lens. This hardware allows for a programmable selection of the wavelength and the illumination design, and provides fast data acquisition as well. Its performance is experimentally demonstrated in the case of ODT with partially coherent illumination providing speckle-free 3D quantitative imaging.

Single Spore Isolation as a Simple and Efficient Technique to obtain fungal pure culture

NASA Astrophysics Data System (ADS)

Noman, E.; Al-Gheethi, AA; Rahman, N. K.; Talip, B.; Mohamed, R.; H, N.; Kadir, O. A.

2018-04-01

The successful identification of fungi by phenotypic methods or molecular technique depends mainly on the using an advanced technique for purifying the isolates. The most efficient is the single spore technique due to the simple requirements and the efficiency in preventing the contamination by yeast, mites or bacteria. The method described in the present work is depends on the using of a light microscope to transfer one spore into a new culture medium. The present work describes a simple and efficient procedure for single spore isolation to purify of fungi recovered from the clinical wastes.

An optical clearing technique for plant tissues allowing deep imaging and compatible with fluorescence microscopy.

PubMed

Warner, Cherish A; Biedrzycki, Meredith L; Jacobs, Samuel S; Wisser, Randall J; Caplan, Jeffrey L; Sherrier, D Janine

2014-12-01

We report on a nondestructive clearing technique that enhances transmission of light through specimens from diverse plant species, opening unique opportunities for microscope-enabled plant research. After clearing, plant organs and thick tissue sections are amenable to deep imaging. The clearing method is compatible with immunocytochemistry techniques and can be used in concert with common fluorescent probes, including widely adopted protein tags such as GFP, which has fluorescence that is preserved during the clearing process. © 2014 American Society of Plant Biologists. All Rights Reserved.

Biological applications of an LCoS-based programmable array microscope (PAM)

NASA Astrophysics Data System (ADS)

Hagen, Guy M.; Caarls, Wouter; Thomas, Martin; Hill, Andrew; Lidke, Keith A.; Rieger, Bernd; Fritsch, Cornelia; van Geest, Bert; Jovin, Thomas M.; Arndt-Jovin, Donna J.

2007-02-01

We report on a new generation, commercial prototype of a programmable array optical sectioning fluorescence microscope (PAM) for rapid, light efficient 3D imaging of living specimens. The stand-alone module, including light source(s) and detector(s), features an innovative optical design and a ferroelectric liquid-crystal-on-silicon (LCoS) spatial light modulator (SLM) instead of the DMD used in the original PAM design. The LCoS PAM (developed in collaboration with Cairn Research, Ltd.) can be attached to a port of a(ny) unmodified fluorescence microscope. The prototype system currently operated at the Max Planck Institute incorporates a 6-position high-intensity LED illuminator, modulated laser and lamp light sources, and an Andor iXon emCCD camera. The module is mounted on an Olympus IX71 inverted microscope with 60-150X objectives with a Prior Scientific x,y, and z high resolution scanning stages. Further enhancements recently include: (i) point- and line-wise spectral resolution and (ii) lifetime imaging (FLIM) in the frequency domain. Multiphoton operation and other nonlinear techniques should be feasible. The capabilities of the PAM are illustrated by several examples demonstrating single molecule as well as lifetime imaging in live cells, and the unique capability to perform photoconversion with arbitrary patterns and high spatial resolution. Using quantum dot coupled ligands we show real-time binding and subsequent trafficking of individual ligand-growth factor receptor complexes on and in live cells with a temporal resolution and sensitivity exceeding those of conventional CLSM systems. The combined use of a blue laser and parallel LED or visible laser sources permits photoactivation and rapid kinetic analysis of cellular processes probed by photoswitchable visible fluorescent proteins such as DRONPA.

Diffracting aperture based differential phase contrast for scanning X-ray microscopy.

PubMed

Kaulich, Burkhard; Polack, Francois; Neuhaeusler, Ulrich; Susini, Jean; di Fabrizio, Enzo; Wilhein, Thomas

2002-10-07

It is demonstrated that in a zone plate based scanning X-ray microscope, used to image low absorbing, heterogeneous matter at a mesoscopic scale, differential phase contrast (DPC) can be implemented without adding any additional optical component to the normal scheme of the microscope. The DPC mode is simply generated by an appropriate positioning and alignment of microscope apertures. Diffraction from the apertures produces a wave front with a non-uniform intensity. The signal recorded by a pinhole photo diode located in the intensity gradient is highly sensitive to phase changes introduced by the specimen to be recorded. The feasibility of this novel DPC technique was proven with the scanning X-ray microscope at the ID21 beamline of the European Synchrotron Radiation facility (ESRF) operated at 6 keV photon energy. We observe a differential phase contrast, similar to Nomarski's differential interference contrast for the light microscope, which results in a tremendous increase in image contrast of up to 20 % when imaging low absorbing specimen.

Quantitatively characterizing the microstructural features of breast ductal carcinoma tissues in different progression stages by Mueller matrix microscope.

PubMed

Dong, Yang; Qi, Ji; He, Honghui; He, Chao; Liu, Shaoxiong; Wu, Jian; Elson, Daniel S; Ma, Hui

2017-08-01

Polarization imaging has been recognized as a potentially powerful technique for probing the microstructural information and optical properties of complex biological specimens. Recently, we have reported a Mueller matrix microscope by adding the polarization state generator and analyzer (PSG and PSA) to a commercial transmission-light microscope, and applied it to differentiate human liver and cervical cancerous tissues with fibrosis. In this paper, we apply the Mueller matrix microscope for quantitative detection of human breast ductal carcinoma samples at different stages. The Mueller matrix polar decomposition and transformation parameters of the breast ductal tissues in different regions and at different stages are calculated and analyzed. For more quantitative comparisons, several widely-used image texture feature parameters are also calculated to characterize the difference in the polarimetric images. The experimental results indicate that the Mueller matrix microscope and the polarization parameters can facilitate the quantitative detection of breast ductal carcinoma tissues at different stages.

Design and performance of an X-ray scanning microscope at the Hard X-ray Nanoprobe beamline of NSLS-II

DOE PAGES

Nazaretski, E.; Yan, H.; Lauer, K.; ...

2017-10-05

A hard X-ray scanning microscope installed at the Hard X-ray Nanoprobe beamline of the National Synchrotron Light Source II has been designed, constructed and commissioned. The microscope relies on a compact, high stiffness, low heat dissipation approach and utilizes two types of nanofocusing optics. It is capable of imaging with ~15 nm × 15 nm spatial resolution using multilayer Laue lenses and 25 nm × 26 nm resolution using zone plates. Fluorescence, diffraction, absorption, differential phase contrast, ptychography and tomography are available as experimental techniques. The microscope is also equipped with a temperature regulation system which allows the temperature ofmore » a sample to be varied in the range between 90 K and 1000 K. The constructed instrument is open for general users and offers its capabilities to the material science, battery research and bioscience communities.« less

Design and performance of an X-ray scanning microscope at the Hard X-ray Nanoprobe beamline of NSLS-II

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

Nazaretski, E.; Yan, H.; Lauer, K.

A hard X-ray scanning microscope installed at the Hard X-ray Nanoprobe beamline of the National Synchrotron Light Source II has been designed, constructed and commissioned. The microscope relies on a compact, high stiffness, low heat dissipation approach and utilizes two types of nanofocusing optics. It is capable of imaging with ~15 nm × 15 nm spatial resolution using multilayer Laue lenses and 25 nm × 26 nm resolution using zone plates. Fluorescence, diffraction, absorption, differential phase contrast, ptychography and tomography are available as experimental techniques. The microscope is also equipped with a temperature regulation system which allows the temperature ofmore » a sample to be varied in the range between 90 K and 1000 K. The constructed instrument is open for general users and offers its capabilities to the material science, battery research and bioscience communities.« less

[Improvement of the microcinematography technic for the study of cell cycles].

PubMed

Gueulette, J; Beauduin, M; Grégoire, V; Van Dorpe, J C; Wambersie, A

1984-10-01

An improvement of time-lapse microcinematography technique is described. It consists in directly printing the time on the microscopical frame, at the moment of the shooting. The time (digital watch), as well as other relevant parameters (temperature etc.) are displayed on a "parameter board", the image of which is encrusted into the microscopical frame by means of an auxiliary two-component lens system. These lenses (current type of microscopical and photographical objectives) are centered on an axis perpendicular to the microscope-camera axis and provide a reduced image of the "parameter board", which is projected on the film edge after deflection by a 45 degree mirror. The latter (aluminized perspex sheet) is located above the photographical eyepiece; it is pierced at the place of the eyepoint in order to give way to the light rays coming out of the cellular culture.

Light field imaging and application analysis in THz

NASA Astrophysics Data System (ADS)

Zhang, Hongfei; Su, Bo; He, Jingsuo; Zhang, Cong; Wu, Yaxiong; Zhang, Shengbo; Zhang, Cunlin

2018-01-01

The light field includes the direction information and location information. Light field imaging can capture the whole light field by single exposure. The four-dimensional light field function model represented by two-plane parameter, which is proposed by Levoy, is adopted in the light field. Acquisition of light field is based on the microlens array, camera array and the mask. We calculate the dates of light-field to synthetize light field image. The processing techniques of light field data include technology of refocusing rendering, technology of synthetic aperture and technology of microscopic imaging. Introducing the technology of light field imaging into THz, the efficiency of 3D imaging is higher than that of conventional THz 3D imaging technology. The advantages compared with visible light field imaging include large depth of field, wide dynamic range and true three-dimensional. It has broad application prospects.

Microgravity Foam Structure and Rheology

NASA Technical Reports Server (NTRS)

Durian, Douglas J.

1997-01-01

To exploit rheological and multiple-light scattering techniques, and ultimately microgravity conditions, in order to quantify and elucidate the unusual elastic character of foams in terms of their underlying microscopic structure and dynamics. Special interest is in determining how this elastic character vanishes, i.e. how the foam melts into a simple viscous liquid, as a function of both increasing liquid content and shear strain rate. The unusual elastic character of foams will be quantified macroscopically by measurement of the shear stress as a function of static shear strain, shear strain rate, and time following a step strain; such data will be analyzed in terms of a yield stress, a static shear modulus, and dynamical time scales. Microscopic information about bubble packing and rearrangement dynamics, from which these macroscopic non-Newtonian properties presumably arise, will be obtained non-invasively by novel multiple-light scattering diagnostics such as Diffusing-Wave Spectroscopy (DWS). Quantitative trends with materials parameters, such as average bubble size, and liquid content, will be sought in order to elucidate the fundamental connection between the microscopic structure and dynamics and the macroscopic rheology.

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

Microscopical and functional aspects of calcium-transport and deposition in terrestrial isopods.

PubMed

Ziegler, Andreas; Fabritius, Helge; Hagedorn, Monica

2005-01-01

Terrestrial isopods (Crustacea) are excellent model organisms to study epithelial calcium-transport and the regulation of biomineralization processes. They molt frequently and resorb cuticular CaCO(3) before the molt to prevent excessive loss of Ca(2+) ions when the old cuticle is shed. The resorbed mineral is stored in CaCO(3) deposits within the ecdysial gap of the first four anterior sternites. After the molt, the deposits are quickly resorbed to mineralise the posterior part of the new cuticle. The deposits contain numerous small spherules composed of an organic matrix and amorphous CaCO(3), which has a high solubility and, therefore, facilitates quick mobilization of Ca(2+) and HCO(3)(-) ions. During the formation and resorption of the deposits large amounts of Ca(2+), HCO(3)(-) and H(+) are transported across the anterior sternal epithelial cells. Within the last years, various light and electron microscopical techniques have been used to characterize the CaCO(3) deposits and the cellular mechanisms involved in biomineralization. The work on the CaCO(3) deposits includes studies on the ultrastructure of the deposits, the sequence of events during deposit formation and dissolution, and the mineral composition of the sternal deposits. The differentiation of the anterior sternal epithelial cells and the mechanisms of epithelial ion transport required for the mineralization and demineralisation of the deposits was studied using various analytical light and electron microscopical techniques including polarized light microscopy, immunocytochemistry, electron microprobe analysis, electron energy loss spectroscopy and electron spectroscopic imaging. Comparative analysis of deposit morphology and the differentiation of the sternal epithelia provide information on the evolution of CaCO(3) deposit formation in relation to the degree of adaptation to terrestrial environments.

Application of principal component analysis to multispectral-multimodal optical image analysis for malaria diagnostics.

PubMed

Omucheni, Dickson L; Kaduki, Kenneth A; Bulimo, Wallace D; Angeyo, Hudson K

2014-12-11

Multispectral imaging microscopy is a novel microscopic technique that integrates spectroscopy with optical imaging to record both spectral and spatial information of a specimen. This enables acquisition of a large and more informative dataset than is achievable in conventional optical microscopy. However, such data are characterized by high signal correlation and are difficult to interpret using univariate data analysis techniques. In this work, the development and application of a novel method which uses principal component analysis (PCA) in the processing of spectral images obtained from a simple multispectral-multimodal imaging microscope to detect Plasmodium parasites in unstained thin blood smear for malaria diagnostics is reported. The optical microscope used in this work has been modified by replacing the broadband light source (tungsten halogen lamp) with a set of light emitting diodes (LEDs) emitting thirteen different wavelengths of monochromatic light in the UV-vis-NIR range. The LEDs are activated sequentially to illuminate same spot of the unstained thin blood smears on glass slides, and grey level images are recorded at each wavelength. PCA was used to perform data dimensionality reduction and to enhance score images for visualization as well as for feature extraction through clusters in score space. Using this approach, haemozoin was uniquely distinguished from haemoglobin in unstained thin blood smears on glass slides and the 590-700 spectral range identified as an important band for optical imaging of haemozoin as a biomarker for malaria diagnosis. This work is of great significance in reducing the time spent on staining malaria specimens and thus drastically reducing diagnosis time duration. The approach has the potential of replacing a trained human eye with a trained computerized vision system for malaria parasite blood screening.

Changes in wood microstructure through progressive stages of decay

Treesearch

W. Wayne Wilcox

1968-01-01

Successive stages of decay in the sapwood of sweetgum (Liquidambar styraciflua L.), a hardwood, and of southern pine (Pinus sp.), a softwood, were observed microscopically. The white-rot fungus. Polyporus versicolor L., and the brown-rot fungus, Poria monticola Murr., were the fungi used or the observations, light microscopy, plus the techniques of polarization and...

A Rubric for Assessing a Student's Ability to Use the Light Microscope

ERIC Educational Resources Information Center

Fitch, Greg K.

2007-01-01

All teachers do assessments. Biology teachers, by grading exams, quizzes, papers, and lab reports, assess mostly "knowledge." An important part of being a modern biologist, however, is the ability to perform certain technical or manual skills (known in the trade as "techniques") such as running gels, pipetting, recording from excitable cells with…

Holographic microscopy for in situ studies of microorganism motility

NASA Astrophysics Data System (ADS)

Nadeau, J.; Hu, S.; Jericho, S.; Lindensmith, C.

2011-12-01

Robust technologies for the detection and identification of microorganisms at low concentrations in complex liquid media are needed for numerous applications: environmental and medical microbiology, food safety, and for the search for microbial life elsewhere in the Solar System. The best current method for microbial enumeration is specific labeling with fluorescent dyes followed by high-resolution light microscopy. However, fluorescent techniques are difficult to use in situ in extreme environments (such as the Arctic and Antarctic or the open ocean) due to the fragility of the instruments and their high power demands. In addition, light microscopic techniques rarely provide insight into microbial motility behaviors. Tracking single cells would provide important insight into the physics of micron-scale motility as well as into key microbial phenomena such as surface attachment and invasiveness. An alternative to traditional light microscopy that is attracting increasing attention is holographic microscopy. Holographic microscopy works by illuminating the object of interest with coherent light from a laser. The light reflected from (or transmitted through) the object is then combined with a coherent reference beam to create an interference pattern that contains the phase and intensity information required to reconstruct a three dimensional image of the object. The interference pattern is recorded on a high resolution detector and can be used to computationally reconstruct a 3D image of the object. The lateral resolution of the image depends upon the wavelength of the light used, the laser power, camera quality, and external noise sources (vibration, stray light, and so forth). Although the principle is simple, technological barriers have prevented wider use of holographic microscopy. Laser sources and CCD cameras with the appropriate properties have only very recently become affordable. In addition, holographic microscopy leads to large data sets that are computationally intensive to reconstruct images from, so the technology to store and process large amounts of data are required. We have successfully deployed a digital in-line holographic microscope in lakes of the Canadian High Arctic and the open ocean. We present characteristic data sets from these experiments, as well as discussing how data acquisition and instrumentation can be improved. A design for a new type of autonomous, submersible holographic microscope incorporating an off-axis reference beam is presented, and future plans for controlled microbe-polymer studies are detailed.

A setup for combined multiphoton laser scanning microscopic and multi-electrode patch clamp experiments on brain slices

NASA Astrophysics Data System (ADS)

Helm, P. Johannes; Reppen, Trond; Heggelund, Paul

2009-02-01

Multi Photon Laser Scanning Microscopy (MPLSM) appears today as one of the most powerful experimental tools in cellular neurophysiology, notably in studies of the functional dynamics of signal processing in single neurons. Simultaneous recording of fluorescence signals at high spatial and temporal resolution and electric signals by means of multi electrode patch clamp techniques have provided new paths for the systematic investigation of neuronal mechanisms. In particular, this approach has opened for direct studies of dendritic signal processing in neurons. We report about a setup optimized for simultaneous electrophysiological multi electrode patch clamp and multi photon laser scanning fluorescence microscopic experiments on brain slices. The microscopic system is based on a modified commercially available confocal scanning laser microscope (CLSM). From a technical and operational point of view, two developments are important: Firstly, in order to reduce the workload for the experimentalist, who in general is forced to concentrate on controlling the electrophysiological parameters during the recordings, a system of shutters has been installed together with dedicated electronic modules protecting the photo detectors against destructive light levels caused by erroneous opening or closing of microscopic light paths by the experimentalist. Secondly, the standard detection unit has been improved by installing the photomultiplier tubes (PMT) in a Peltier cooled thermal box shielding the detector from both room temperature and distortions caused by external electromagnetic fields. The electrophysiological system is based on an industrial standard multi patch clamp unit ergonomically arranged around the microscope stage. The electrophysiological and scanning processes can be time coordinated by standard trigger electronics.

L-Phenylalanine functionalized silver nanoparticles: Photocatalytic and nonlinear optical applications

NASA Astrophysics Data System (ADS)

Nidya, M.; Umadevi, M.; Sankar, Pranitha; Philip, Reji; Rajkumar, Beulah J. M.

2015-04-01

An extensive study on the behavior of L-Phenylalanine capped silver nanoparticles (Phe-Ag NPs) in the aqueous phase and in a sol-gel thin film showed different UV/Vis, Transmission Electron Microscope (TEM), Dynamic Light Scattering and Zeta potential profiles. Scanning Electron Microscope (SEM) images of the samples in the sol gel film showed Ag embedded in the SiO2 matrix. Surface Enhanced Raman Spectra (SERS) confirmed that both in the aqueous media and in the sol gel film, the attachment of Phe to the Ag NP surface was through the benzene ring, with the sol-gel film showing a better enhancement. Photocatalytic degradation of crystal violet was measured spectrophotometrically using Phe-Ag NPs as a nanocatalyst under visible light illumination. Intensity-dependent nonlinear optical absorption of Phe-Ag measured using the open aperture Z-scan technique revealed that the material is an efficient optical limiter with potential applications.

Linear polarization-discriminatory state inverter fabricated by oblique angle deposition.

PubMed

Park, Yong Jun; Sobahan, K M A; Kim, Jin Joo; Hwangbo, Chang Kwon

2009-06-22

In this paper, we report a linear polarization-discriminatory state inverter made of three-layer sculpture thin film fabricated by oblique angle deposition technique. The first and third layers are quarter-wave plates of zigzag structure and the middle of them is a circular Bragg reflector of left-handed helical structure. It is found that the normal incidence of P-polarized light on this polarization-discriminatory state inverter becomes the S-polarized light at output, while the incident S-polarized light of wavelength lying in the Bragg regime is reflected. The microstructure of the linear polarization-discriminatory state inverter is also investigated by using a scanning electron microscope.

Dependency of subcellular reactions during PDT on the metabolic state of cell cultures probed by different microscopic techniques

NASA Astrophysics Data System (ADS)

Rueck, Angelika C.; Schneckenburger, Herbert; Strauss, Wolfgang S. L.; Gschwend, Michael H.; Beck, Gerd C.; Kunzi-Rapp, Karin; Steiner, Rudolf W.

1994-02-01

Various microscopic techniques were used to study the dependency of photodynamically induced subcellular reactions on the metabolic state of cell cultures. TPPS4 and AlS2-3Pc were incubated in RR 1022 epithelial cells with varying cell density. To attain almost isolated cells (low cell density) or confluent growing cells (high cell density) 25 cells/mm2 or 500 cells/mm2 were seeded, respectively. Low cell density irradiation with blue light led to a change in the initial cytoplasmatic fluorescence pattern. For both sensitizers, TPPS4 as well as AlS2-3, a fluorescence relocalization and fluorescence intensity increase could be detected, moreover in the case of TPPS4 a fluorescence formation in the nucleus and nucleoli were detected. In contrast, for confluent growing cells no redistribution was observed.

Correlative fractography: combining scanning electron microscopy and light microscopes for qualitative and quantitative analysis of fracture surfaces.

PubMed

Hein, Luis Rogerio de Oliveira; de Oliveira, José Alberto; de Campos, Kamila Amato

2013-04-01

Correlative fractography is a new expression proposed here to describe a new method for the association between scanning electron microscopy (SEM) and light microscopy (LM) for the qualitative and quantitative analysis of fracture surfaces. This article presents a new method involving the fusion of one elevation map obtained by extended depth from focus reconstruction from LM with exactly the same area by SEM and associated techniques, as X-ray mapping. The true topographic information is perfectly associated to local fracture mechanisms with this new technique, presented here as an alternative to stereo-pair reconstruction for the investigation of fractured components. The great advantage of this technique resides in the possibility of combining any imaging methods associated with LM and SEM for the same observed field from fracture surface.

Identification of mycobacterium tuberculosis in sputum smear slide using automatic scanning microscope

NASA Astrophysics Data System (ADS)

Rulaningtyas, Riries; Suksmono, Andriyan B.; Mengko, Tati L. R.; Saptawati, Putri

2015-04-01

Sputum smear observation has an important role in tuberculosis (TB) disease diagnosis, because it needs accurate identification to avoid high errors diagnosis. In development countries, sputum smear slide observation is commonly done with conventional light microscope from Ziehl-Neelsen stained tissue and it doesn't need high cost to maintain the microscope. The clinicians do manual screening process for sputum smear slide which is time consuming and needs highly training to detect the presence of TB bacilli (mycobacterium tuberculosis) accurately, especially for negative slide and slide with less number of TB bacilli. For helping the clinicians, we propose automatic scanning microscope with automatic identification of TB bacilli. The designed system modified the field movement of light microscope with stepper motor which was controlled by microcontroller. Every sputum smear field was captured by camera. After that some image processing techniques were done for the sputum smear images. The color threshold was used for background subtraction with hue canal in HSV color space. Sobel edge detection algorithm was used for TB bacilli image segmentation. We used feature extraction based on shape for bacilli analyzing and then neural network classified TB bacilli or not. The results indicated identification of TB bacilli that we have done worked well and detected TB bacilli accurately in sputum smear slide with normal staining, but not worked well in over staining and less staining tissue slide. However, overall the designed system can help the clinicians in sputum smear observation becomes more easily.

Microgravity foam structure and rheology

NASA Technical Reports Server (NTRS)

Durian, Douglas J.; Gopal, Anthony D.

1994-01-01

Our long-range objective is to establish the fundamental interrelationship between the microscopic structure and dynamics of foams and their macroscopic stability and rheology. Foam structure and dynamics are to be measured directly and noninvasively through the use and development of novel multiple light scattering techniques such as diffusing-wave spectroscopy (DWS). Foam rheology is to be measured in a custom rheometer which allows simultaneous optical access for multiple light drainage of liquid from in between gas bubbles as the liquid:gas volume fraction in increased towards the rigidity-loss transition.

Effects of spaceflight in the adductor longus muscle of rats flown in the Soviet Biosatellite COSMOS 2044. A study employing neural cell adhesion molecule (N-CAM) immunocytochemistry and conventional morphological techniques (light and electron microscopy)

NASA Technical Reports Server (NTRS)

D'Amelio, F.; Daunton, N. G.

1992-01-01

The effects of spaceflight upon the "slow" muscle adductor longus were examined in rats flown in the Soviet Biosatellite COSMOS 2044. The techniques employed included standard methods for light microscopy, neural cell adhesion molecule (N-CAM) immunocytochemistry and electron microscopy. Light microscopic observations revealed myofiber atrophy and segmental necrosis accompanied by cellular infiltrates composed of macrophages, leukocytes and mononuclear cells. Neural cell adhesion molecule immunoreactivity (N-CAM-IR) was seen on the myofiber surface and in regenerating myofibers. Ultrastructural alterations included Z band streaming, disorganization of myofibrillar architecture, sarcoplasmic degradation, extensive segmental necrosis with apparent preservation of the basement membrane, degenerative phenomena of the capillary endothelium and cellular invasion of necrotic areas. Regenerating myofibers were identified by the presence of increased amounts of ribosomal aggregates and chains of polyribosomes associated with myofilaments. The principal electron microscopic changes of the neuromuscular junctions showed axon terminals with a decrease or absence of synaptic vesicles replaced by microtubules and neurofilaments, degeneration of axon terminals, vacant axonal spaces and changes suggestive of axonal sprouting. The present observations suggest that alterations such as myofibrillar disruption and necrosis, muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight.

New stroboscopic light source and technique for intraoperative retinal fluorescein angiography during penetrating keratoplasty

NASA Astrophysics Data System (ADS)

Krueger, Ronald R.; Morales, Ronald B.; Chong, Lawrence P.; Smith, Ronald E.

1994-06-01

We report the development of a new stroboscopic light source system and technique for performing intraoperative fluorescein angiography during penetrating keratoplasty for aphakic or pseudophakic bullous keratopathy. A controllable pulse xenon light source system with a fiber optic endoilluminator probe is used to perform high-quality intraoperative fluorescein angiography during penetrating keratoplasty in pigmented rabbits and human subjects. Following corneal trephination and extraction of the intraocular lens, a temporary Cobo keratoprosthesis is secured while a 20-gauge endoilluminator is inserted into the vitreous cavity through a limbal incision. The endoilluminator is advanced to a retinal illumination area of approximately 3 DD and 10% fluorescein is injected intravenously. A microscope camera coupled to a 50:50 beamsplitter photographs the passage of fluorescein dye while the surgeon maintains an unaltered view through the operating microscope. Angiograms through a keratoprosthesis show excellent contrast and resolution, comparable to standard fluorescein angiography. Fine peripapillary vessels are seen reproducibly and with great detail in the rabbits. All the phases of retinal angiography can be seen, including arteriolar constriction and capillary nonperfusion in one of four human subjects examined. High quality intraoperative fluorescein angiography can be performed in patients undergoing penetrating keratoplasty for aphakic/ pseudophakic bullous keratopathy. With this technology, preexisting retinal disorders such as cystoid macular edema might be identified in the perioperative setting allowing for important management decisions to be made intraoperatively.

Identifying mitosis deep in tissue using dynamic light scattering fluctuation spectroscopy

NASA Astrophysics Data System (ADS)

An, Ran; Jeong, Kwan; Turek, John; Nolte, David

2012-03-01

In the cell cycle, mitosis is the most dramatic phase, especially in Telophase and Cytokinesis. For single cells and cell monolayer, there are precise microscopic studies of mitosis, while for 3-D tissue such as tumor spheroids the light signal is obscured by the high background of diffusely scattered light. Therefore, the mitosis phase cannot be detected deep inside 3-D tissue using conventional microscopic techniques. In this work, we detect mitosis in living tissue using Tissue Dynamic Imaging (TDI). We trace depth-gated dynamic speckles from a tumor spheroid (up to 1mm in diameter) using coherence-gated digital holography imaging. Frequency-versus-time spectrograms depend on specific types of perturbation such as cell shape change, membrane undulation and cell organelles movements. By using these spectral responses as functional finger prints, we can identify mitosis events from different voxels at a specified depth inside tumor spheroids. By performing B-scans of the tumor spheroid, we generate 3-D mitosis maps (or movies) for the entire tumor spheroids. We show that for healthy tumor spheroids, the mitosis events only happen within the proliferating shell. We also compare results when anti-cancer drugs are applied to arrest, release and synchronize mitosis. This shows the application of TDI for drug screening. The technique can identify and monitor complex motilities inside 3-D tissue with a strong potential for drug diagnosis and developmental biology studies.

Assessment of incomplete clipping of aneurysms intraoperatively by a near-infrared indocyanine green-video angiography (Niicg-Va) integrated microscope.

PubMed

Imizu, S; Kato, Y; Sangli, A; Oguri, D; Sano, H

2008-08-01

The objective of this article was to assess the clinical use and the completeness of clipping with total occlusion of the aneurysmal lumen, real-time assessment of vascular patency in the parent, branching and perforating vessels, intraoperative assessment of blood flow, image quality, spatial resolution and clinical value in difficult aneurysms using near infrared indocyanine green video angiography integrated on to an operative Pentero neurosurgical microscope (Carl Zeiss, Oberkochen Germany). Thirteen patients with aneurysms were operated upon. An infrared camera with near infrared technology was adapted on to the OPMI Pentero microscope with a special filter and infrared excitation light to illuminate the operating field which was designed to allow passage of the near infrared light required for excitation of indocyanine green (ICG) which was used as the intravascular marker. The intravascular fluorescence was imaged with a video camera attached to the microscope. ICG fluorescence (700-850 nm) from a modified microscope light source on to the surgical field and passage of ICG fluorescence (780-950 nm) from the surgical field, back into the optical path of the microscope was used to detect the completeness of aneurysmal clipping Incomplete clipping in three patients (1 female and 2 males) with unruptured complicated aneurysms was detected using indocyanine green video angiography. There were no adverse effects after injection of indocyanine green. The completeness of clipping was inadequately detected by Doppler ultrasound miniprobe and rigid endoscopy and was thus complemented by indocyanine green video angiography. The operative microscope-integrated ICG video angiography as a new intraoperative method for detecting vascular flow, was found to be quick, reliable, cost-effective and possibly a substitute or adjunct for Doppler ultrasonography or intraoperative DSA, which is presently the gold standard. The simplicity of the method, the speed with which the investigation can be performed, the quality of the images, and the outcome of surgical procedures have all reduced the need for angiography. This technique may be useful during routine aneurysm surgery as an independent form of angiography and/or as an adjunct to intraoperative or postoperative DSA.

Visualizing 3-D microscopic specimens

NASA Astrophysics Data System (ADS)

Forsgren, Per-Ola; Majlof, Lars L.

1992-06-01

The confocal microscope can be used in a vast number of fields and applications to gather more information than is possible with a regular light microscope, in particular about depth. Compared to other three-dimensional imaging devices such as CAT, NMR, and PET, the variations of the objects studied are larger and not known from macroscopic dissections. It is therefore important to have several complementary ways of displaying the gathered information. We present a system where the user can choose display techniques such as extended focus, depth coding, solid surface modeling, maximum intensity and other techniques, some of which may be combined. A graphical user interface provides easy and direct control of all input parameters. Motion and stereo are available options. Many three- dimensional imaging devices give recordings where one dimension has different resolution and sampling than the other two which requires interpolation to obtain correct geometry. We have evaluated algorithms with interpolation in object space and in projection space. There are many ways to simplify the geometrical transformations to gain performance. We present results of some ways to simplify the calculations.

Looking at tardigrades in a new light: using epifluorescence to interpret structure.

PubMed

Perry, E S; Miller, W R; Lindsay, S

2015-02-01

The use of epifluorescence microscopy coupled with ultraviolet (UV) autofluorescence is suggested as a means to view and interpret tardigrade structures. Endogenous fluorochromes are a known component of tardigrade cuticle, claws and bucco-pharyngeal apparatus. By imaging the autofluorescence from tardigrades, it is possible to document these structures in detail, including the subdivisions and boundaries of echiniscid (heterotardigrade) plates and the nature and spatial relationships of the texture (pores, granules, papillae and tubercles) on the various plates. This allows the determination of taxonomic features not easily seen with other microscopic techniques. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

Microcircuit failure analysis using the SEM. [Scanning Electron Microscopes

NASA Technical Reports Server (NTRS)

Nicolas, D. P.

1974-01-01

The scanning electron microscope adds a new dimension to the knowledge that can be obtained from a failed microcircuit. When used with conventional techniques, SEM assists and clarifies the analysis, but it does not replace light microscopy. The most advantageous features for microcircuit analysis are long working distances and great depth of field. Manufacturer related failure modes of microcircuits are metallization defects, poor bonding, surface and particle contamination, and design and fabrication faults. User related failure modes are caused by abuse, such as overstress. The Physics of Failure Procedure followed by the Astrionics Laboratory in failure analysis is described, which is designed to obtain maximum information available from each step.

Development of a scanning time of flight microscope and its application to the study of charge transport in phase separated structured organic semiconductors

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

Paul, Sanjoy; Ellman, Brett, E-mail: bellman@kent.edu; Singh, Gautam

We describe a tool for studying the two-dimensional spatial variation in electronic properties of organic semiconductors: the scanning time-of-flight microscope (STOFm). The STOFm simultaneously measures the transmittance of polarized light and time-of-flight current transients with a pixel size <30 μm, making it especially valuable for studies of the correlations of structure with charge generation and transport in liquid crystalline organic semiconductors (LC OSCs). Adapting a previously developed photopolymerization technique, we characterize the instrument using patterned samples of a LC OSC bounded by a non-semiconducting polymer matrix.

Symposium N: Materials and Devices for Thermal-to-Electric Energy Conversion

DTIC Science & Technology

2010-08-24

X - ray diffraction, transmission electron microscopy, scanning electron microscopy, and dynamic light scattering. Thermal conductivity measurements...SEM), X - ray diffraction (XRD) measurements as well as Raman spectroscopy. The results from these techniques indicate a clear modification...was examined by using scanning electron microscope (SEM; HITACHI S-4500 model) attached with an energy dispersive x - ray spectroscopy. The electrical

Cathodoluminescence in the scanning transmission electron microscope.

PubMed

Kociak, M; Zagonel, L F

2017-05-01

Cathodoluminescence (CL) is a powerful tool for the investigation of optical properties of materials. In recent years, its combination with scanning transmission electron microscopy (STEM) has demonstrated great success in unveiling new physics in the field of plasmonics and quantum emitters. Most of these results were not imaginable even twenty years ago, due to conceptual and technical limitations. The purpose of this review is to present the recent advances that broke these limitations, and the new possibilities offered by the modern STEM-CL technique. We first introduce the different STEM-CL operating modes and the technical specificities in STEM-CL instrumentation. Two main classes of optical excitations, namely the coherent one (typically plasmons) and the incoherent one (typically light emission from quantum emitters) are investigated with STEM-CL. For these two main classes, we describe both the physics of light production under electron beam irradiation and the physical basis for interpreting STEM-CL experiments. We then compare STEM-CL with its better known sister techniques: scanning electron microscope CL, photoluminescence, and electron energy-loss spectroscopy. We finish by comprehensively reviewing recent STEM-CL applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Cathodoluminescence in the scanning transmission electron microscope.

PubMed

Kociak, M; Zagonel, L F

2016-12-19

Cathodoluminescence (CL) is a powerful tool for the investigation of optical properties of materials. In recent years, its combination with scanning transmission electron microscopy (STEM) has demonstrated great success in unveiling new physics in the field of plasmonics and quantum emitters. Most of these results were not imaginable even twenty years ago, due to conceptual and technical limitations. The purpose of this review is to present the recent advances that broke these limitations, and the new possibilities offered by the modern STEM-CL technique. We first introduce the different STEM-CL operating modes and the technical specificities in STEM-CL instrumentation. Two main classes of optical excitations, namely the coherent one (typically plasmons) and the incoherent one (typically light emission from quantum emitters) are investigated with STEM-CL. For these two main classes, we describe both the physics of light production under electron beam irradiation and the physical basis for interpreting STEM-CL experiments. We then compare STEM-CL with its better known sister techniques: scanning electron microscope CL, photoluminescence, and electron energy-loss spectroscopy. We finish by comprehensively reviewing recent STEM-CL applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Universal method for creating optically active nanostructures on layered materials

NASA Astrophysics Data System (ADS)

Kidd, Tim; He, Rui; Stollenwerk, Andrew; Oshea, Aaron; Beck, Ben; Spurgeon, Kyle; Gu, Genda

2014-03-01

We report a new method for the creating of nanostructures using a scanning electron microscope. Residual organic molecules on the surface of layered materials can be excited by electron beam radiation to burrow into the open spaces between the layers of these materials, and then are broken down further to form photoluminescent carbon nanoclusters. Surface characterization by atomic force microscopy shows the surface is nearly undamaged at the molecular level by this process, and a lack of nanostructure formation in non-layered materials confirms that the structures are created by sub-surface incorporation. The presence of carbon nanoclusters was determined by Raman Spectroscopy and photoluminescence in the visible light range. The nanostructures are react strongly to visible light, making them readily apparent using an optical microscope even for features measuring only a few nanometers tall. This technique can be used on apparently any layered material, with successful results on dichalcogenides, topological insulators, graphite, and high temperature copper oxide superconductors. This technique can create patterned nanostructures with vertical resolution at the nanometer scale and lateral resolution of tens of nanometers depending on beam spot size. This work is funded by University of Northern Iowa, NSF #DMR-1206530, and DOE #DE-AC02-98CH10886.

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

Guo, Wei, E-mail: wguo2@ncsu.edu; Kirste, Ronny; Bryan, Zachary

Enhanced light extraction efficiency was demonstrated on nanostructure patterned GaN and AlGaN/AlN Multiple-Quantum-Well (MQW) structures using mass production techniques including natural lithography and interference lithography with feature size as small as 100 nm. Periodic nanostructures showed higher light extraction efficiency and modified emission profile compared to non-periodic structures based on integral reflection and angular-resolved transmission measurement. Light extraction mechanism of macroscopic and microscopic nanopatterning is discussed, and the advantage of using periodic nanostructure patterning is provided. An enhanced photoluminescence emission intensity was observed on nanostructure patterned AlGaN/AlN MQW compared to as-grown structure, demonstrating a large-scale and mass-producible pathway to higher lightmore » extraction efficiency in deep-ultra-violet light-emitting diodes.« less

Surface topography characterization using 3D stereoscopic reconstruction of SEM images

NASA Astrophysics Data System (ADS)

Vedantha Krishna, Amogh; Flys, Olena; Reddy, Vijeth V.; Rosén, B. G.

2018-06-01

A major drawback of the optical microscope is its limitation to resolve finer details. Many microscopes have been developed to overcome the limitations set by the diffraction of visible light. The scanning electron microscope (SEM) is one such alternative: it uses electrons for imaging, which have much smaller wavelength than photons. As a result high magnification with superior image resolution can be achieved. However, SEM generates 2D images which provide limited data for surface measurements and analysis. Often many research areas require the knowledge of 3D structures as they contribute to a comprehensive understanding of microstructure by allowing effective measurements and qualitative visualization of the samples under study. For this reason, stereo photogrammetry technique is employed to convert SEM images into 3D measurable data. This paper aims to utilize a stereoscopic reconstruction technique as a reliable method for characterization of surface topography. Reconstructed results from SEM images are compared with coherence scanning interferometer (CSI) results obtained by measuring a roughness reference standard sample. This paper presents a method to select the most robust/consistent surface texture parameters that are insensitive to the uncertainties involved in the reconstruction technique itself. Results from the two-stereoscopic reconstruction algorithms are also documented in this paper.

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.

Portable, battery-operated, fluorescence field microscope for the developing world

NASA Astrophysics Data System (ADS)

Miller, Andrew R.; Davis, Gregory; Pierce, Mark; Oden, Z. Maria; Richards-Kortum, Rebecca

2010-02-01

In many areas of the world, current methods for diagnosis of infectious diseases such as malaria and tuberculosis involve microscopic evaluation of a patient specimen. Advances in fluorescence microscopy can improve diagnostic sensitivity and reduce time and expertise necessary to interpret diagnostic results. However, modern research-grade microscopes are neither available nor appropriate for use in many settings in the developing world. To address this need, we designed, fabricated, and tested a portable, battery-powered, bright field and fluorescence inverted field microscope, optimized for infrastructural constraints of the developing world. We characterized an initial prototype constructed with rapidprototyping techniques, which utilized low-cost, over-the-counter components such as a battery-powered LED flashlight as the light source. The microscope exhibited suitable spatial resolution (0.8 μm) in fluorescence mode to resolve M. tuberculosis bacilli. In bright field mode, malaria parasites were resolvable at 1000x magnification. The initial prototype cost 480 USD and we estimate that the microscope can be manufactured for 230 USD. While future studies are planned to evaluate ease-of-use and reliability, our current system serves as a proof of concept that combined fluorescence and bright field microscopy is possible in a low-cost and portable system.

Spectro-microscopy of living plant cells.

PubMed

Harter, Klaus; Meixner, Alfred J; Schleifenbaum, Frank

2012-01-01

Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techniques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on recent developments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context. The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassinosteroide receptor BRI1 in the plasma membrane of living plant cells. Moreover, the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime (FLT). Furthermore, a new spectro-microscopic technique, fluorescence intensity decay shape analysis microscopy (FIDSAM), has been developed. FIDSAM is capable of imaging low-expressed fluorophore-tagged proteins at high spatial resolution and precludes the misinterpretation of autofluorescence artifacts. In addition, FIDSAM provides a very effective and sensitive tool on the basis of Förster resonance energy transfer (FRET) for the qualitative and quantitative determination of protein-protein interaction. Finally, we report on the quantitative analysis of the photosystem I and II (PSI/PSII) ratio in the chloroplasts of living Arabidopsis plants at room temperature, using high-resolution, spatially resolved fluorescence spectroscopy. With this technique, it was not only possible to measure PSI/PSII ratios, but also to demonstrate the differential competence of wild-type and carbohydrate-deficient plants to adapt the PSI/PSII ratio to different light conditions. In summary, the information content of standard microscopic images is extended by several dimensions by the use of spectro-microscopic approaches. Therefore, novel cell physiological and molecular topics can be addressed and valuable insights into molecular and subcellular processes can be obtained in living plants.

Far-infrared Beamline at the Canadian Light Source

NASA Astrophysics Data System (ADS)

Zhao, Jianbao; Billinghurst, Brant

2017-06-01

Far-infrared is a particularly useful technique for studies on lattice modes as they generally appear in the Far-infrared region. Far-infrared is also an important tool for gathering information on the electrical transport properties of metallic materials and the band gap of semiconductors. This poster will describe the horizontal microscope that has recently been built in the Far-infrared beamline at the Canadian Light Source Inc. (CLS). This microscope is specially designed for high-pressure Far-infrared absorbance and reflectance spectroscopic studies. The numerical aperture (0.5) and the long working distance (82.1 mm) in the microscope are good fits for Diamond Anvil Cell (DAC). The spectra are recorded using liquid helium cooled Si bolometer or Ge:Cu detector. The pressure in the DAC can be determined by using the fluorescence spectrometer available onsite. The Far-infrared beamline at CLS is a state-of-the-art synchrotron facility, offering significantly more brightness than conventional sources. Because of the high brightness of the synchrotron radiation, we can obtain the Far-infrared reflectance/absorbance spectra on the small samples with more throughput than with a conventional source. The Far-infrared beamline is open to users through peer review.

An integrated single- and two-photon non-diffracting light-sheet microscope

NASA Astrophysics Data System (ADS)

Lau, Sze Cheung; Chiu, Hoi Chun; Zhao, Luwei; Zhao, Teng; Loy, M. M. T.; Du, Shengwang

2018-04-01

We describe a fluorescence optical microscope with both single-photon and two-photon non-diffracting light-sheet excitations for large volume imaging. With a special design to accommodate two different wavelength ranges (visible: 400-700 nm and near infrared: 800-1200 nm), we combine the line-Bessel sheet (LBS, for single-photon excitation) and the scanning Bessel beam (SBB, for two-photon excitation) light sheet together in a single microscope setup. For a transparent thin sample where the scattering can be ignored, the LBS single-photon excitation is the optimal imaging solution. When the light scattering becomes significant for a deep-cell or deep-tissue imaging, we use SBB light-sheet two-photon excitation with a longer wavelength. We achieved nearly identical lateral/axial resolution of about 350/270 nm for both imagings. This integrated light-sheet microscope may have a wide application for live-cell and live-tissue three-dimensional high-speed imaging.

Development of Low-Cost Inverted Microscope to Detect Early Growth of Mycobacterium tuberculosis in MODS Culture

PubMed Central

Zimic, Mirko; Velazco, Abner; Comina, Germán; Coronel, Jorge; Fuentes, Patricia; Luna, Carmen G.; Sheen, Patricia; Gilman, Robert H.; Moore, David A. J.

2010-01-01

Background The microscopic observation drug susceptibility (MODS) assay for rapid, low-cost detection of tuberculosis and multidrug resistant tuberculosis depends upon visualization of the characteristic cording colonies of Mycobacterium tuberculosis in liquid media. This has conventionally required an inverted light microscope in order to inspect the MODS culture plates from below. Few tuberculosis laboratories have this item and the capital cost of $5,000 for a high-end microscope could be a significant obstacle to MODS roll-out. Methodology We hypothesized that the precise definition provided by costly high-specification inverted light microscopes might not be necessary for pattern recognition. Significance In this work we describe the development of a low-cost artesenal inverted microscope that can operate in both a standard or digital mode to effectively replace the expensive commercial inverted light microscope, and an integrated system that could permit a local and remote diagnosis of tuberculosis. PMID:20351778

Macular photostress and visual experience between microscope and intracameral illumination during cataract surgery.

PubMed

Seo, Hyejin; Nam, Dong Heun; Lee, Jong Yeon; Park, Su Jin; Kim, Yu Jeong; Kim, Seong-Woo; Chung, Tae-Young; Inoue, Makoto; Kim, Terry

2018-02-01

To evaluate macular photostress and visual experience between coaxial microscope illumination versus oblique intracameral illumination during cataract surgery. Gachon University Gil Hospital, Incheon, South Korea. Prospective case series. Consecutive patients who had cataract surgery using microscope illumination and intracameral illumination were included. The patients were asked to complete a questionnaire (seeing strong lights, feeling photophobia, feeling startled (fright) when seeing lights, seeing any colors, seeing any instruments or surgical procedures, and estimating intraoperative visual function) designed to describe their cataract surgery experience. The images projected on the retina of the model eye (rear view) with artificial opaque fragments in the anterior chamber during simulating cataract surgery were compared between the 2 illumination types. Sixty patients completed the questionnaire. Scores for strong lights, photophobia, fright, and color perception were significantly higher with microscope illumination than with intracameral illumination (all P < .001). More patients preferred the intracameral illumination (45 [75.0%]) to the microscope illumination (13 [21.7%]). In the rear-view images created in a model eye, only the bright microscope light in the center was seen without any lens image in the microscope illumination. However, in the intracameral illumination, the less bright light from the light pipe in the periphery and the lens fragments were seen more clearly. In a view of the patients' visual experience, oblique intracameral illumination caused less subjective photostress and was preferred over coaxial microscope illumination. Objective findings from the model-eye experiment correlated to the result of visual experience. Copyright © 2018 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Assessing various Infrared (IR) microscopic imaging techniques for post-mortem interval evaluation of human skeletal remains.

PubMed

Woess, Claudia; Unterberger, Seraphin Hubert; Roider, Clemens; Ritsch-Marte, Monika; Pemberger, Nadin; Cemper-Kiesslich, Jan; Hatzer-Grubwieser, Petra; Parson, Walther; Pallua, Johannes Dominikus

2017-01-01

Due to the influence of many environmental processes, a precise determination of the post-mortem interval (PMI) of skeletal remains is known to be very complicated. Although methods for the investigation of the PMI exist, there still remains much room for improvement. In this study the applicability of infrared (IR) microscopic imaging techniques such as reflection-, ATR- and Raman- microscopic imaging for the estimation of the PMI of human skeletal remains was tested. PMI specific features were identified and visualized by overlaying IR imaging data with morphological tissue structures obtained using light microscopy to differentiate between forensic and archaeological bone samples. ATR and reflection spectra revealed that a more prominent peak at 1042 cm-1 (an indicator for bone mineralization) was observable in archeological bone material when compared with forensic samples. Moreover, in the case of the archaeological bone material, a reduction in the levels of phospholipids, proteins, nucleic acid sugars, complex carbohydrates as well as amorphous or fully hydrated sugars was detectable at (reciprocal wavelengths/energies) between 3000 cm-1 to 2800 cm-1. Raman spectra illustrated a similar picture with less ν2PO43-at 450 cm-1 and ν4PO43- from 590 cm-1 to 584 cm-1, amide III at 1272 cm-1 and protein CH2 deformation at 1446 cm-1 in archeological bone material/samples/sources. A semi-quantitative determination of various distributions of biomolecules by chemi-maps of reflection- and ATR- methods revealed that there were less carbohydrates and complex carbohydrates as well as amorphous or fully hydrated sugars in archaeological samples compared with forensic bone samples. Raman- microscopic imaging data showed a reduction in B-type carbonate and protein α-helices after a PMI of 3 years. The calculated mineral content ratio and the organic to mineral ratio displayed that the mineral content ratio increases, while the organic to mineral ratio decreases with time. Cluster-analyses of data from Raman microscopic imaging reconstructed histo-anatomical features in comparison to the light microscopic image and finally, by application of principal component analyses (PCA), it was possible to see a clear distinction between forensic and archaeological bone samples. Hence, the spectral characterization of inorganic and organic compounds by the afore mentioned techniques, followed by analyses such as multivariate imaging analysis (MIAs) and principal component analyses (PCA), appear to be suitable for the post mortem interval (PMI) estimation of human skeletal remains.

Assessing various Infrared (IR) microscopic imaging techniques for post-mortem interval evaluation of human skeletal remains

PubMed Central

Roider, Clemens; Ritsch-Marte, Monika; Pemberger, Nadin; Cemper-Kiesslich, Jan; Hatzer-Grubwieser, Petra; Parson, Walther; Pallua, Johannes Dominikus

2017-01-01

Due to the influence of many environmental processes, a precise determination of the post-mortem interval (PMI) of skeletal remains is known to be very complicated. Although methods for the investigation of the PMI exist, there still remains much room for improvement. In this study the applicability of infrared (IR) microscopic imaging techniques such as reflection-, ATR- and Raman- microscopic imaging for the estimation of the PMI of human skeletal remains was tested. PMI specific features were identified and visualized by overlaying IR imaging data with morphological tissue structures obtained using light microscopy to differentiate between forensic and archaeological bone samples. ATR and reflection spectra revealed that a more prominent peak at 1042 cm-1 (an indicator for bone mineralization) was observable in archeological bone material when compared with forensic samples. Moreover, in the case of the archaeological bone material, a reduction in the levels of phospholipids, proteins, nucleic acid sugars, complex carbohydrates as well as amorphous or fully hydrated sugars was detectable at (reciprocal wavelengths/energies) between 3000 cm-1 to 2800 cm-1. Raman spectra illustrated a similar picture with less ν2PO43−at 450 cm-1 and ν4PO43− from 590 cm-1 to 584 cm-1, amide III at 1272 cm-1 and protein CH2 deformation at 1446 cm-1 in archeological bone material/samples/sources. A semi-quantitative determination of various distributions of biomolecules by chemi-maps of reflection- and ATR- methods revealed that there were less carbohydrates and complex carbohydrates as well as amorphous or fully hydrated sugars in archaeological samples compared with forensic bone samples. Raman- microscopic imaging data showed a reduction in B-type carbonate and protein α-helices after a PMI of 3 years. The calculated mineral content ratio and the organic to mineral ratio displayed that the mineral content ratio increases, while the organic to mineral ratio decreases with time. Cluster-analyses of data from Raman microscopic imaging reconstructed histo-anatomical features in comparison to the light microscopic image and finally, by application of principal component analyses (PCA), it was possible to see a clear distinction between forensic and archaeological bone samples. Hence, the spectral characterization of inorganic and organic compounds by the afore mentioned techniques, followed by analyses such as multivariate imaging analysis (MIAs) and principal component analyses (PCA), appear to be suitable for the post mortem interval (PMI) estimation of human skeletal remains. PMID:28334006

The Direct-Indirect Technique for Composite Restorations Revisited.

PubMed

Ritter, André V; Fahl, Newton; Vargas, Marcos; Maia, Rodrigo R

2017-06-01

In the direct-indirect composite technique, composite is applied to a nonretentive tooth preparation (eg, a noncarious cervical lesion or a veneer/inlay/onlay preparation) without any bonding agent, sculpted to a primary anatomic form, and light-cured. The partially polymerized restoration is then removed from the preparation and finished and tempered extraorally chairside. The finished inlay is bonded to the preparation using a resin-based luting agent. Advantages of this technique include enhanced physical and mechanical properties afforded by the extraoral chairside tempering process because of increased monomer conversion, and greater operator control over the final marginal adaptation, surface finishing and polishing, and anatomy of the restoration, given that these elements are defined outside of the patient's mouth. The direct-indirect approach also affords enhanced gingival health and patient comfort. This article presents a clinical case in which the direct-indirect composite technique was used to restore three noncarious cervical lesions on the same quadrant on an adult patient. Clinical steps and tips for success are offered. The authors also present scanning electron microscope and atomic force microscope images showing the excellent marginal fit obtained with the direct-indirect composite technique.

Automated system for characterization and classification of malaria-infected stages using light microscopic images of thin blood smears.

PubMed

Das, D K; Maiti, A K; Chakraborty, C

2015-03-01

In this paper, we propose a comprehensive image characterization cum classification framework for malaria-infected stage detection using microscopic images of thin blood smears. The methodology mainly includes microscopic imaging of Leishman stained blood slides, noise reduction and illumination correction, erythrocyte segmentation, feature selection followed by machine classification. Amongst three-image segmentation algorithms (namely, rule-based, Chan-Vese-based and marker-controlled watershed methods), marker-controlled watershed technique provides better boundary detection of erythrocytes specially in overlapping situations. Microscopic features at intensity, texture and morphology levels are extracted to discriminate infected and noninfected erythrocytes. In order to achieve subgroup of potential features, feature selection techniques, namely, F-statistic and information gain criteria are considered here for ranking. Finally, five different classifiers, namely, Naive Bayes, multilayer perceptron neural network, logistic regression, classification and regression tree (CART), RBF neural network have been trained and tested by 888 erythrocytes (infected and noninfected) for each features' subset. Performance evaluation of the proposed methodology shows that multilayer perceptron network provides higher accuracy for malaria-infected erythrocytes recognition and infected stage classification. Results show that top 90 features ranked by F-statistic (specificity: 98.64%, sensitivity: 100%, PPV: 99.73% and overall accuracy: 96.84%) and top 60 features ranked by information gain provides better results (specificity: 97.29%, sensitivity: 100%, PPV: 99.46% and overall accuracy: 96.73%) for malaria-infected stage classification. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

Method for nanoscale spatial registration of scanning probes with substrates and surfaces

NASA Technical Reports Server (NTRS)

Wade, Lawrence A. (Inventor)

2010-01-01

Embodiments in accordance with the present invention relate to methods and apparatuses for aligning a scanning probe used to pattern a substrate, by comparing the position of the probe to a reference location or spot on the substrate. A first light beam is focused on a surface of the substrate as a spatial reference point. A second light beam then illuminates the scanning probe being used for patterning. An optical microscope images both the focused light beam, and a diffraction pattern, shadow, or light backscattered by the illuminated scanning probe tip of a scanning probe microscope (SPM), which is typically the tip of the scanning probe on an atomic force microscope (AFM). Alignment of the scanning probe tip relative to the mark is then determined by visual observation of the microscope image. This alignment process may be repeated to allow for modification or changing of the scanning probe microscope tip.

Microscope use in clinical veterinary practice and potential implications for veterinary school curricula.

PubMed

Stewart, Sherry M; Dowers, Kristy L; Cerda, Jacey R; Schoenfeld-Tacher, Regina M; Kogan, Lori R

2014-01-01

Microscopy (skill of using a microscope) and the concepts of cytology (study of cells) and histology (study of tissues) are most often taught in professional veterinary medicine programs through the traditional method of glass slides and light microscopes. Several limiting factors in veterinary training programs are encouraging educators to explore innovative options for teaching microscopy skills and the concepts of cytology and histology. An anonymous online survey was administered through the Colorado Veterinary Medical Association to Colorado veterinarians working in private practice. It was designed to assess their current usage of microscopes for cytological and histological evaluation of specimens and their perceptions of microscope use in their veterinary education. The first part of the survey was answered by 183 veterinarians, with 104 indicating they had an onsite diagnostic lab. Analysis pertaining to the use of the microscope in practice and in veterinary programs was conducted on this subset. Most respondents felt the amount of time spent in the curriculum using a microscope was just right for basic microscope use and using the microscope for viewing and learning about normal and abnormal histological sections and clinical cytology. Participants felt more emphasis could be placed on clinical and diagnostic cytology. Study results suggest that practicing veterinarians frequently use microscopes for a wide variety of cytological diagnostics. However, only two respondents indicated they prepared samples for histological evaluation. Veterinary schools should consider these results against the backdrop of pressure to implement innovative teaching techniques to meet the changing needs of the profession.

Optical spectroscopies diagnose cancer

NASA Astrophysics Data System (ADS)

Alfano, Robert R.; Das, Bidyut B.; Glassman, Wenling S.; Pradhan, Asima; Tang, Gui C.

1992-02-01

Today's medical professional is looking beyond the conventional procedures of X-rays, nuclear radiation, magnetic resonance, chemical analysis, and ultrasound to diagnose diseases ranging from cancer to heart ailments. In view of the possible dangerous side effects of X-rays and nuclear radiation, a need exists for novel techniques in disease detection that can either eliminate or reduce their use in examinations. For more than half a century, fluorescence, absorption, and light scattering spectroscopies have been widely used as probes to acquire fundamental knowledge about various physical, chemical, and biological processes. Light may offer alternatives to X-rays and nuclear approaches, and in some cases is non-invasive. Optical spectroscopy and laser technology may offer techniques for the detection and characterization of physical and chemical changes that occur in diseased tissue on a microscopic level.

Improvement in the luminous efficiency of MEH-PPV based light emitting diodes using zinc oxide nanorods grown by the electrochemical deposition technique on ITO substrates

NASA Astrophysics Data System (ADS)

Gupta, Rohini B.; Kumar, Jitender; Madhwal, Devinder; Singh, Inderpreet; Kaur, I.; Bhardwaj, L. M.; Nagpal, S.; Bhatnagar, P. K.; Mathur, P. C.

2011-07-01

Zinc oxide (ZnO) nanorods grown by the electrochemical technique have been used to enhance the luminance of poly[2-methoxy-5-(2'-ethylhexoxy)-1,4-phenylenevinylene] (MEH-PPV)-based polymer light-emitting diodes. The luminance of the device with ZnO nanorods is found to increase by more than two times as compared with the device without ZnO nanorods. The diameter of the nanorods used in device fabrication was ~145 nm. The size of the nanorods was estimated from field emission scanning electron microscope images. Optical and structural characterizations of the nanorods were also performed by using absorption, photoluminescence and x-ray diffraction, confirming the formation of ZnO nanorods.

Highly sensitive mode mapping of whispering-gallery modes by scanning thermocouple-probe microscopy.

PubMed

Klein, Angela E; Schmidt, Carsten; Liebsch, Mattes; Janunts, Norik; Dobynde, Mikhail; Tünnermann, Andreas; Pertsch, Thomas

2014-03-01

We propose a method for mapping optical near-fields with the help of a thermocouple scanning-probe microscope tip. As the tip scans the sample surface, its apex is heated by light absorption, generating a thermovoltage. The thermovoltage map represents the intensity distribution of light at the sample surface. The measurement technique has been employed to map optical whispering-gallery modes in fused silica microdisk resonators operating at near-infrared wavelengths. The method could potentially be employed for near-field imaging of a variety of systems in the near-infrared and visible spectral range.

A light field microscope imaging spectrometer based on the microlens array

NASA Astrophysics Data System (ADS)

Yao, Yu-jia; Xu, Feng; Xia, Yin-xiang

2017-10-01

A new light field spectrometry microscope imaging system, which was composed by microscope objective, microlens array and spectrometry system was designed in this paper. 5-D information (4-D light field and 1-D spectrometer) of the sample could be captured by the snapshot system in only one exposure, avoiding the motion blur and aberration caused by the scanning imaging process of the traditional imaging spectrometry. Microscope objective had been used as the former group while microlens array used as the posterior group. The optical design of the system was simulated by Zemax, the parameter matching condition between microscope objective and microlens array was discussed significantly during the simulation process. The result simulated in the image plane was analyzed and discussed.

Determination of morphological parameters of biological cells by analysis of scattered-light distributions

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

Burger, D.E.

1979-11-01

The extraction of morphological parameters from biological cells by analysis of light-scatter patterns is described. A light-scattering measurement system has been designed and constructed that allows one to visually examine and photographically record biological cells or cell models and measure the light-scatter pattern of an individual cell or cell model. Using a laser or conventional illumination, the imaging system consists of a modified microscope with a 35 mm camera attached to record the cell image or light-scatter pattern. Models of biological cells were fabricated. The dynamic range and angular distributions of light scattered from these models was compared to calculatedmore » distributions. Spectrum analysis techniques applied on the light-scatter data give the sought after morphological cell parameters. These results compared favorably to shape parameters of the fabricated cell models confirming the mathematical model procedure. For nucleated biological material, correct nuclear and cell eccentricity as well as the nuclear and cytoplasmic diameters were determined. A method for comparing the flow equivalent of nuclear and cytoplasmic size to the actual dimensions is shown. This light-scattering experiment provides baseline information for automated cytology. In its present application, it involves correlating average size as measured in flow cytology to the actual dimensions determined from this technique. (ERB)« less

Intraoperative Fluorescence Cerebral Angiography by Laser Surgical Microscopy: Comparison With Xenon Microscopy and Simultaneous Observation of Cerebral Blood Flow and Surrounding Structures.

PubMed

Ito, Yuhei; Suzuki, Kyouichi; Ichikawa, Tsuyoshi; Watanabe, Yoichi; Sato, Taku; Sakuma, Jun; Saito, Kiyoshi

2018-06-12

Laser surgical microscopes should enable uniform illumination of the operative field, and require less luminous energy compared with existing xenon surgical microscopes. To examine the utility of laser illumination in fluorescence cerebral angiography. Fluorescein sodium (fluorescein) was used as a fluorescent dye. We first compared the clarity of cerebral blood flow images collected by fluorescence angiography between the laser illumination and xenon illumination methods. We then assessed use of the laser illuminator for simultaneous observation of blood flow and surrounding structures during fluorescence angiography. Furthermore, the study was designed to evaluate usefulness of the thus determined excitation light in clinical cases. Fluorescence angiography using blue light laser for excitation provided higher clarity and contrast blood flow images compared with using blue light generated from a xenon lamp. Further, illumination with excitation light consisting of a combination of 3 types of laser (higher level of blue light, no green light, and lower level of red light) enabled both blood flow and surrounding structures to be observed through the microscope directly by the surgeon. Laser-illuminated fluorescence angiography provides high clarity and contrast images of cerebral blood flow. Further, a laser providing strong blue light and weak red light for excitation light enables simultaneous visual observation of fluorescent blood flow and surrounding structures by the surgeon using a surgical microscope. Overall, these data suggest that laser surgical microscopes are useful for both ordinary operative manipulations and fluorescence angiography.

Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains.

PubMed

Müllenbroich, M Caroline; Silvestri, Ludovico; Onofri, Leonardo; Costantini, Irene; Hoff, Marcel Van't; Sacconi, Leonardo; Iannello, Giulio; Pavone, Francesco S

2015-10-01

Comprehensive mapping and quantification of neuronal projections in the central nervous system requires high-throughput imaging of large volumes with microscopic resolution. To this end, we have developed a confocal light-sheet microscope that has been optimized for three-dimensional (3-D) imaging of structurally intact clarified whole-mount mouse brains. We describe the optical and electromechanical arrangement of the microscope and give details on the organization of the microscope management software. The software orchestrates all components of the microscope, coordinates critical timing and synchronization, and has been written in a versatile and modular structure using the LabVIEW language. It can easily be adapted and integrated to other microscope systems and has been made freely available to the light-sheet community. The tremendous amount of data routinely generated by light-sheet microscopy further requires novel strategies for data handling and storage. To complete the full imaging pipeline of our high-throughput microscope, we further elaborate on big data management from streaming of raw images up to stitching of 3-D datasets. The mesoscale neuroanatomy imaged at micron-scale resolution in those datasets allows characterization and quantification of neuronal projections in unsectioned mouse brains.

Microscopic theory of linear light scattering from mesoscopic media and in near-field optics.

PubMed

Keller, Ole

2005-08-01

On the basis of quantum mechanical response theory a microscopic propagator theory of linear light scattering from mesoscopic systems is presented. The central integral equation problem is transferred to a matrix equation problem by discretization in transitions between pairs of (many-body) energy eigenstates. The local-field calculation which appears from this approach is valid down to the microscopic region. Previous theories based on the (macroscopic) dielectric constant concept make use of spatial (geometrical) discretization and cannot in general be trusted on the mesoscopic length scale. The present theory can be applied to light scattering studies in near-field optics. After a brief discussion of the macroscopic integral equation problem a microscopic potential description of the scattering process is established. In combination with the use of microscopic electromagnetic propagators the formalism allows one to make contact to the macroscopic theory of light scattering and to the spatial photon localization problem. The quantum structure of the microscopic conductivity response tensor enables one to establish a clear physical picture of the origin of local-field phenomena in mesoscopic and near-field optics. The Huygens scalar propagator formalism is revisited and its generality in microscopic physics pointed out.

Phase resolved and coherence gated en face reflection imaging of multilayered embryonal carcinoma cells

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Fukami, Tadashi; Iwai, Hidenao; Yamashita, Yutaka

2012-03-01

Embryonal carcinoma (EC) cells, which are cell lines derived from teratocarcinomas, have characteristics in common with stem cells and differentiate into many kinds of functional cells. Similar to embryonic stem (ES) cells, undifferentiated EC cells form multi-layered spheroids. In order to visualize the three-dimensional structure of multilayered EC cells without labeling, we employed full-field interference microscopy with the aid of a low-coherence quantitative phase microscope, which is a reflection-type interference microscope employing the digital holographic technique with a low-coherent light source. Owing to the low-coherency of the light-source (halogen lamp), only the light reflected from reflective surface at a specific sectioning height generates an interference image on the CCD camera. P19CL6 EC cells, derived from mouse teratocarcinomas, formed spheroids that are about 50 to 200 micrometers in diameter. Since the height of each cell is around 10 micrometers, it is assumed that each spheroid has 5 to 20 cell layers. The P19CL6 spheroids were imaged in an upright configuration and the horizontally sectioned reflection images of the sample were obtained by sequentially and vertically scanning the zero-path-length height. Our results show the threedimensional structure of the spheroids, in which plasma and nuclear membranes were distinguishably imaged. The results imply that our technique is further capable of imaging induced pluripotent stem (iPS) cells for the assessment of cell properties including their pluripotency.

Experiment K-7-18: Effects of Spaceflight in the Muscle Adductor Longus of Rats Flown in the Soviet Biosatellite Cosmos 2044. Part 1; A Study Employing Neural Cell Adhesion Molecules (N-CAM) Immunocytochemistry and Conventional Morphological Techniques (Light and Electron Microscopy)

NASA Technical Reports Server (NTRS)

Daunton, N. G.; DAmelio, F.; Wu, L.; Ilyina-Kakueva, E. I.; Krasnov, I. B.; Hyde, T. M.; Sigworth, S. K.

1994-01-01

The effects of spaceflight upon the 'slow' muscle adductor longus was examined in rats flown in the Soviet Biosatellite COSMOS 2044. Three groups - synchronous, vivarium and basal served as controls. The techniques employed included standard methods for light microscopy, N-CAM immunocytochemistry and electron microscopy. Light microscopic observations revealed myofiber atrophy, contraction bands and segmental necrosis accompanied by cellular infiltrates composed of macrophages, leucocytes and mononuclear cells. N-CAM immunoreactivity was seen (N-CAM-IR) on the myofiber surface, satellite cells and in regenerating myofibers reminiscent of myotubes. Ultrastructural alterations included Z band streaming, disorganization of myofibrillar architecture, sarcoplasmic degradation, extensive segmental necrosis with preservation of the basement membrane, degenerative phenomena of the capillary endothelium and cellular invasion of necrotic areas. Regenerating myofibers were identified by the presence of increased amounts of ribosomal aggregates and chains of polyribosomes associated with myofilaments that displayed varied distributive patterns. The principal electron microscopic changes of the neuromuscular junctions consisted of a decrease or absence of synaptic vesicles, degeneration of axon terminals, increased number of microtubules, vacant axonal spaces and axonal sprouting. The present observations indicate that major alterations such as myofibrillar disruption and necrosis, muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight.

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest

NASA Astrophysics Data System (ADS)

Dahlberg, Peter D.; Boughter, Christopher T.; Faruk, Nabil F.; Hong, Lu; Koh, Young Hoon; Reyer, Matthew A.; Shaiber, Alon; Sherani, Aiman; Zhang, Jiacheng; Jureller, Justin E.; Hammond, Adam T.

2016-11-01

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH3NH3PbBr3 perovskites and measure differences between nanocrystal films and micron scale crystals.

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest.

PubMed

Dahlberg, Peter D; Boughter, Christopher T; Faruk, Nabil F; Hong, Lu; Koh, Young Hoon; Reyer, Matthew A; Shaiber, Alon; Sherani, Aiman; Zhang, Jiacheng; Jureller, Justin E; Hammond, Adam T

2016-11-01

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH 3 NH 3 PbBr 3 perovskites and measure differences between nanocrystal films and micron scale crystals.

Shack-Hartmann reflective micro profilometer

NASA Astrophysics Data System (ADS)

Gong, Hai; Soloviev, Oleg; Verhaegen, Michel; Vdovin, Gleb

2018-01-01

We present a quantitative phase imaging microscope based on a Shack-Hartmann sensor, that directly reconstructs the optical path difference (OPD) in reflective mode. Comparing with the holographic or interferometric methods, the SH technique needs no reference beam in the setup, which simplifies the system. With a preregistered reference, the OPD image can be reconstructed from a single shot. Also, the method has a rather relaxed requirement on the illumination coherence, thus a cheap light source such as a LED is feasible in the setup. In our previous research, we have successfully verified that a conventional transmissive microscope can be transformed into an optical path difference microscope by using a Shack-Hartmann wavefront sensor under incoherent illumination. The key condition is that the numerical aperture of illumination should be smaller than the numerical aperture of imaging lens. This approach is also applicable to characterization of reflective and slightly scattering surfaces.

Fast parallel 3D profilometer with DMD technology

NASA Astrophysics Data System (ADS)

Hou, Wenmei; Zhang, Yunbo

2011-12-01

Confocal microscope has been a powerful tool for three-dimensional profile analysis. Single mode confocal microscope is limited by scanning speed. This paper presents a 3D profilometer prototype of parallel confocal microscope based on DMD (Digital Micromirror Device). In this system the DMD takes the place of Nipkow Disk which is a classical parallel scanning scheme to realize parallel lateral scanning technique. Operated with certain pattern, the DMD generates a virtual pinholes array which separates the light into multi-beams. The key parameters that affect the measurement (pinhole size and the lateral scanning distance) can be configured conveniently by different patterns sent to DMD chip. To avoid disturbance between two virtual pinholes working at the same time, a scanning strategy is adopted. Depth response curve both axial and abaxial were extract. Measurement experiments have been carried out on silicon structured sample, and axial resolution of 55nm is achieved.

Epifluorescence light collection for multiphoton microscopic endoscopy

NASA Astrophysics Data System (ADS)

Brown, Christopher M.; Rivera, David R.; Xu, Chris; Webb, Watt W.

2011-03-01

Multiphoton microscopic endoscopy (MPM-E) is a promising medical in vivo diagnostic imaging technique because it captures intrinsic fluorescence and second harmonic generation signals to reveal anatomical and histological information about disease states in tissue. However, maximizing light collection from multiphoton endoscopes remains a challenge: weak nonlinear emissions from endogenous structures, miniature optics, large imaging depths, and light scattering in tissue all hamper light collection. The quantity of light that may be collected using a dual-clad fiber system from scattering phantoms that mimic the properties of the in vivo environment is measured. In this experiment, 800nm excitation light from a Ti:Sapphire laser is dispersion compensated and focused through a SM800 optical fiber and lens system into the tissue phantom. Emission light from the phantom passes through the lens system, reflects off the dichroic and is then collected by a second optical fiber actuated by a micromanipulator. The lateral position of the collection fiber varies, measuring the distribution of emitted light 2000μm on either side of the focal point reimaged to the object plane. This spatial collection measurement is performed at depths up to 200μm from the phantom surface. The tissue phantoms are composed of a 15.8 μM fluorescein solution mixed with microspheres, approximating the scattering properties of human bladder and dermis tissue. Results show that commercially available dual-clad optical fibers collect more than 47% of the total emission returning to the object plane from both phantoms. Based on these results, initial MPM-E devices will image the surface of epithelial tissues.

Influence of tray rigidity and impression technique on accuracy of polyvinyl siloxane impressions.

PubMed

Hoyos, Alex; Soderholm, Karl-Johan

2011-01-01

The aim of this study was to determine how tray rigidity and impression technique affect the accuracy of polyvinyl siloxane impressions. Disposable plastic trays and metallic Rim-Lock trays were used in combination with a heavy/light body technique or with two different putty-wash techniques. For each technique-tray combination, 10 impressions were made of a master cast with two steel abutments representing the mandibular right first premolar and second molar, between which a steel rod was placed at the ridge level. Each steel abutment had five marks, while the steel rod in between the two abutments had only one mark, which served as a reference point. With a universal measuring microscope, the x-, y-, and z-coordinates were recorded for each mark on the master cast and impressions. The distances between the different marks and the reference point on the impressions were calculated and compared with those of the master cast. All techniques used with the plastic trays had distances that were significantly different from the master cast (P < .05), while for the metal trays, it was only the heavy/light body technique that resulted in distances that were significantly different from the master cast (P < .05). Plastic trays produced less accurate impressions than metal trays. When metal trays were used, putty-based impressions were dimensionally better than heavy/light body impressions.

Optical filters for wavelength selection in fluorescence instrumentation.

PubMed

Erdogan, Turan

2011-04-01

Fluorescence imaging and analysis techniques have become ubiquitous in life science research, and they are poised to play an equally vital role in in vitro diagnostics (IVD) in the future. Optical filters are crucial for nearly all fluorescence microscopes and instruments, not only to provide the obvious function of spectral control, but also to ensure the highest possible detection sensitivity and imaging resolution. Filters make it possible for the sample to "see" light within only the absorption band, and the detector to "see" light within only the emission band. Without filters, the detector would not be able to distinguish the desired fluorescence from scattered excitation light and autofluorescence from the sample, substrate, and other optics in the system. Today the vast majority of fluorescence instruments, including the widely popular fluorescence microscope, use thin-film interference filters to control the spectra of the excitation and emission light. Hence, this unit emphasizes thin-film filters. After briefly introducing different types of thin-film filters and how they are made, the unit describes in detail different optical filter configurations in fluorescence instruments, including both single-color and multicolor imaging systems. Several key properties of thin-film filters, which can significantly affect optical system performance, are then described. In the final section, tunable optical filters are also addressed in a relative comparison.

Light, electron microscopic and immunohistochemical study of the effect of low-dose aspirin during the proestrus phase on rat endometrium in the preimplantation period.

PubMed

Ateş, Utku; Baka, Meral; Turgut, Mehmet; Uyanikgil, Yiğit; Ulker, Sibel; Yilmaz, Ozlem; Tavmergen, Erol; Yurtseven, Mine

2007-04-01

To evaluate structural alterations in rat endometrium at preimplantation following treatment with aspirin beginning from proestrus by light microscopy, electron microscopy and immunohistochemical techniques. Twenty rats were divided into control (n = 10) and experimental (n = 10) groups. Experimental rats were treated with low-dose aspirin daily (2 mg/kg/day) during estrus, beginning from the proestrus phase, mated at end of cycle and treated with aspirin. Untreated pregnant rats were the control group. Rats in both groups were sacrificed at the 84th pregnancy hour; the uterus was rapidly removed and dissected free of surrounding adipose tissue. Uteri specimens from nonpregnant rats were transferred into fixative solution and processed for light, electron microscopic and immunohistochemical study. Light and electron microscopy of endometrium from control rats conformed to mid-diestrus phase; endometrial histology of the aspirin-treated group conformed to late diestrus phase. The endometrial layer was significantly thicker in the aspirin-treated group compared to the untreated control group (p <0.001). No significant difference was found in vessel number between groups. Staining with alphaV integrin was more dense in the aspirin-treated group. Based on histologic findings, we suggest low-dose aspirin has positive effects on preparing endometrium before implantation.

Measurement of anchoring coefficient of homeotropically aligned nematic liquid crystal using a polarizing optical microscope in reflective mode

NASA Astrophysics Data System (ADS)

Baek, Sang-In; Kim, Sung-Jo; Kim, Jong-Hyun

2015-09-01

Although the homeotropic alignment of liquid crystals is widely used in LCD TVs, no easy method exists to measure its anchoring coefficient. In this study, we propose an easy and convenient measurement technique in which a polarizing optical microscope is used in the reflective mode with an objective lens having a low depth of focus. All measurements focus on the reflection of light near the interface between the liquid crystal and alignment layer. The change in the reflected light is measured by applying an electric field. We model the response of the director of the liquid crystal to the electric field and, thus, the change in reflectance. By adjusting the extrapolation length in the calculation, we match the experimental and calculated results and obtain the anchoring coefficient. In our experiment, the extrapolation lengths were 0.31 ± 0.04 μm, 0.32 ± 0.08 μm, and 0.23 ± 0.05 μm for lecithin, AL-64168, and SE-5662, respectively.

Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase. II. Light-microscopic application.

PubMed

Beier, K; Fahimi, H D

1987-01-01

The feasibility of the application of a television-based image analyzer, the Texture Analysis System (TAS, Leitz Wetzlar, FRG) in conjunction with a light microscope for morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained with the alkaline-DAB method for localization of catalase and semithin (0.25 and 1 micron) sections of plastic-embedded material were examined under an oil immersion objective. The TAS detected the peroxisomal profiles selectively and determined their morphometric parameters automatically. The same parameters were obtained also by morphometric analysis of electron micrographs from the same material. The volume density of peroxisomes determined by TAS in semithin sections of normal liver, after correction for section thickness, is quite close to the corresponding value obtained by morphometry of electron micrographs. The difference is approximately 20%. In animals treated with the hypolipidemic drug bezafibrate, which causes proliferation of peroxisomes, TAS detected readily the increase in volume density of peroxisomes in semithin sections. In comparison with electron microscopy, however, the light-microscopic approach seems to underestimate the proliferation. The lower resolution of the light microscope and overlapping of neighbouring particles in relatively thick sections used for light-microscopic analysis may account for the differences. The present study has demonstrated the usefulness of automatic image analysis in conjunction with selective cytochemical staining of peroxisomes for morphometry of this organelle in rat liver. The light-microscopic approach is not only faster but is also extremely economical by obviating the use of an electron microscope.

Microgravity Foam Structure and Rheology

NASA Technical Reports Server (NTRS)

Durian, Douglas J.

1996-01-01

The objective of this research was to exploit rheological and multiple-light scattering techniques, and ultimately microgravity conditions, in order to quantify and elucidate the unusual elastic character of foams in terms of their underlying microscopic structure and dynamics. Special interest was in determining how this elastic character vanishes, i.e. how the foam melts into a simple viscous liquid, as a function of both increasing liquid content and shear strain rate.

Connexin composition in apposed gap junction hemiplaques revealed by matched double-replica freeze-fracture replica immunogold labeling.

PubMed

Rash, John E; Kamasawa, Naomi; Davidson, Kimberly G V; Yasumura, Thomas; Pereda, Alberto E; Nagy, James I

2012-06-01

Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of the membrane "sidedness" and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons.

Utility and safety of a novel surgical microscope laser light source

PubMed Central

Bakhit, Mudathir S.; Suzuki, Kyouichi; Sakuma, Jun; Fujii, Masazumi; Murakami, Yuta; Ito, Yuhei; Sugano, Tetsuo; Saito, Kiyoshi

2018-01-01

Objective Tissue injuries caused by the thermal effects of xenon light microscopes have previously been reported. Due to this, the development of a safe microscope light source became a necessity. A newly developed laser light source is evaluated regarding its effectiveness and safety as an alternative to conventional xenon light source. Methods We developed and tested a new laser light source for surgical microscopes. Four experiments were conducted to compare xenon and laser lights: 1) visual luminance comparison, 2) luminous and light chromaticity measurements, 3) examination and analysis of visual fatigue, and 4) comparison of focal temperature elevation due to light source illumination using porcine muscle samples. Results Results revealed that the laser light could be used at a lower illumination value than the xenon light (p < 0.01). There was no significant difference in visual fatigue status between the laser light and the xenon light. The laser light was superior to the xenon light regarding luminous intensity and color chromaticity. The focal temperature elevation of the muscle samples was significantly higher when irradiated with xenon light in vitro than with laser light (p < 0.01). Conclusion The newly developed laser light source is more efficient and safer than a conventional xenon light source. It lacks harmful ultraviolet waves, has a longer lifespan, a lower focal temperature than that of other light sources, a wide range of brightness and color production, and improved safety for the user’s vision. Further clinical trials are necessary to validate the impact of this new light source on the patient’s outcome and prognosis. PMID:29390016

AccessScope project: Accessible light microscope for users with upper limb mobility or visual impairments.

PubMed

Mansoor, Awais; Ahmed, Wamiq M; Samarapungavan, Ala; Cirillo, John; Schwarte, David; Robinson, J Paul; Duerstock, Bradley S

2010-01-01

A web-based application was developed to remotely view slide specimens and control all functions of a research-level light microscopy workstation, called AccessScope. Students and scientists with upper limb mobility and visual impairments are often unable to use a light microscope by themselves and must depend on others in its operation. Users with upper limb mobility impairments and low vision were recruited to assist in the design process of the AccessScope personal computer (PC) user interface. Participants with these disabilities were evaluated in their ability to use AccessScope to perform microscopical tasks. AccessScope usage was compared with inspecting prescanned slide images by grading participants' identification and understanding of histological features and knowledge of microscope operation. With AccessScope subjects were able to independently perform common light microscopy functions through an Internet browser by employing different PC pointing devices or accessibility software according to individual abilities. Subjects answered more histology and microscope usage questions correctly after first participating in an AccessScope test session. AccessScope allowed users with upper limb or visual impairments to successfully perform light microscopy without assistance. This unprecedented capability is crucial for students and scientists with disabilities to perform laboratory coursework or microscope-based research and pursue science, technology, engineering, and mathematics fields.

A smartphone-based chip-scale microscope using ambient illumination.

PubMed

Lee, Seung Ah; Yang, Changhuei

2014-08-21

Portable chip-scale microscopy devices can potentially address various imaging needs in mobile healthcare and environmental monitoring. Here, we demonstrate the adaptation of a smartphone's camera to function as a compact lensless microscope. Unlike other chip-scale microscopy schemes, this method uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is based on the shadow imaging technique where the sample is placed on the surface of the image sensor, which captures direct shadow images under illumination. To improve the image resolution beyond the pixel size, we perform pixel super-resolution reconstruction with multiple images at different angles of illumination, which are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. The lensless imaging scheme allows for sub-micron resolution imaging over an ultra-wide field-of-view (FOV). Image acquisition and reconstruction are performed on the device using a custom-built Android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.

A smartphone-based chip-scale microscope using ambient illumination

PubMed Central

Lee, Seung Ah; Yang, Changhuei

2014-01-01

Portable chip-scale microscopy devices can potentially address various imaging needs in mobile healthcare and environmental monitoring. Here, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope. Unlike other chip-scale microscopy schemes, this method uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is based on the shadow imaging technique where the sample is placed on the surface of the image sensor, which captures direct shadow images under illumination. To improve the imaging resolution beyond the pixel size, we perform pixel super-resolution reconstruction with multiple images at different angles of illumination, which are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. The lensless imaging scheme allows for sub-micron resolution imaging over an ultra-wide field-of-view (FOV). Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system. PMID:24964209

LC-lens array with light field algorithm for 3D biomedical applications

NASA Astrophysics Data System (ADS)

Huang, Yi-Pai; Hsieh, Po-Yuan; Hassanfiroozi, Amir; Martinez, Manuel; Javidi, Bahram; Chu, Chao-Yu; Hsuan, Yun; Chu, Wen-Chun

2016-03-01

In this paper, liquid crystal lens (LC-lens) array was utilized in 3D bio-medical applications including 3D endoscope and light field microscope. Comparing with conventional plastic lens array, which was usually placed in 3D endoscope or light field microscope system to record image disparity, our LC-lens array has higher flexibility of electrically changing its focal length. By using LC-lens array, the working distance and image quality of 3D endoscope and microscope could be enhanced. Furthermore, the 2D/3D switching ability could be achieved if we turn off/on the electrical power on LClens array. In 3D endoscope case, a hexagonal micro LC-lens array with 350um diameter was placed at the front end of a 1mm diameter endoscope. With applying electric field on LC-lens array, the 3D specimen would be recorded as from seven micro-cameras with different disparity. We could calculate 3D construction of specimen with those micro images. In the other hand, if we turn off the electric field on LC-lens array, the conventional high resolution 2D endoscope image would be recorded. In light field microscope case, the LC-lens array was placed in front of the CMOS sensor. The main purpose of LC-lens array is to extend the refocusing distance of light field microscope, which is usually very narrow in focused light field microscope system, by montaging many light field images sequentially focusing on different depth. With adjusting focal length of LC-lens array from 2.4mm to 2.9mm, the refocusing distance was extended from 1mm to 11.3mm. Moreover, we could use a LC wedge to electrically shift the optics axis and increase the resolution of light field.

A light and scanning electron microscopic evaluation of electro-discharge-compacted porous titanium implants in rabbit tibia.

PubMed

Drummond, J F; Dominici, J T; Sammon, P J; Okazaki, K; Geissler, R; Lifland, M I; Anderson, S A; Renshaw, W

1995-01-01

This study used light and scanning electron microscopic (SEM) histomorphometric methods to quantitate the rate of osseointegration of totally porous titanium alloy (Ti-6Al-4V) implants prepared by a novel fabrication technique--electrodischarge compaction (EDC). EDC was used to fuse 150-250-micrometer spherical titanium alloy beads into 4 X 6 mm cylindrical implants through application of a 300-microsecond pulse of high-voltage/high-current density. Two sterilized implants were surgically placed into each tibia of 20 New Zealand white rabbits and left in situ for periods corresponding to 2, 4, 8, 12, and 24 weeks. At each time point, 4 rabbits were humanely killed, and the implants with surrounding bone were removed, fixed, and sectioned for light and SEM studies. The degree of osseointegration was quantitated by means of a True Grid Digitizing Pad and Jandel Scan Version 3.9 software on an IBM PS/2 computer. The total pore area occupied by bone was divided by the total pore area available for bone ingrowth, and a Bone Ingrowth Factor (BIF) was calculated as a percent. The light microscopic results showed BIFs of 4% at week 2, 47% at week 4, 62% at week 8, 84% at week 12, and greater than 90% at week 24. The SEM results showed BIFs of 5% at week 2, 34% at week 4, 69% at week 8, 75% at week 12, and in excess of 90% at week 24. The results of this study show that EDC implants are biocompatible and support rapid osseointegration in the rabbit tibia and suggest that, after additional studies, they may be suitable for use as dental implants in humans.

A comparative analysis of microscopic alterations in modern and ancient undecalcified and decalcified dry bones.

PubMed

Caruso, Valentina; Cummaudo, Marco; Maderna, Emanuela; Cappella, Annalisa; Caudullo, Giorgio; Scarpulla, Valentina; Cattaneo, Cristina

2018-02-01

The present study aims to evaluate the preservation of the microstructure of skeletal remains collected from four different known burial sites (archaeological and contemporary). Histological analysis on undecalcified and decalcified thin sections was performed in order to assess which of the two techniques is more affected by taphonomic insults. A histological analysis was performed on both undecalcified and decalcified thin sections of 40 long bones and the degree of diagenetic change was evaluated using transmitted and polarized light microscopy according to the Oxford Histological Index (OHI). In order to test the optical behavior of bone tissue, thin sections were observed by polarized light microscopy and the intensity of birefringence was evaluated. The more ancient samples are generally characterized by a low OHI (0-1) with extensive microscopic focal destruction; recent samples exhibited a better preservation of bone micromorphology. When comparing undecalcified to decalcified thin sections, the latter showed an amelioration in the conservation of microscopic structure. As regards the birefringence, it was very low in all the undecalcified thin sections, whereas decalcification process seems to improve its visibility. The preservation of the bone microscopic structure appears to be influenced not only by age, but also by the burial context. Undecalcified bones appear to be more affected by taphonomical alterations, probably because of the thickness of the thin sections; on the contrary, decalcified thin sections proved to be able to tackle this issue allowing a better reading of the bone tissue. © 2017 Wiley Periodicals, Inc.

Note: Laser beam scanning using a ferroelectric liquid crystal spatial light modulator

NASA Astrophysics Data System (ADS)

Das, Abhijit; Boruah, Bosanta R.

2014-04-01

In this work we describe laser beam scanning using a ferroelectric liquid crystal spatial light modulator. Commercially available ferroelectric liquid crystal spatial light modulators are capable of displaying 85 colored images in 1 s using a time dithering technique. Each colored image, in fact, comprises 24 single bit (black and white) images displayed sequentially. We have used each single bit image to write a binary phase hologram. For a collimated laser beam incident on the hologram, one of the diffracted beams can be made to travel along a user defined direction. We have constructed a beam scanner employing the above arrangement and demonstrated its use to scan a single laser beam in a laser scanning optical sectioning microscope setup.

Machine learning approach for automated screening of malaria parasite using light microscopic images.

PubMed

Das, Dev Kumar; Ghosh, Madhumala; Pal, Mallika; Maiti, Asok K; Chakraborty, Chandan

2013-02-01

The aim of this paper is to address the development of computer assisted malaria parasite characterization and classification using machine learning approach based on light microscopic images of peripheral blood smears. In doing this, microscopic image acquisition from stained slides, illumination correction and noise reduction, erythrocyte segmentation, feature extraction, feature selection and finally classification of different stages of malaria (Plasmodium vivax and Plasmodium falciparum) have been investigated. The erythrocytes are segmented using marker controlled watershed transformation and subsequently total ninety six features describing shape-size and texture of erythrocytes are extracted in respect to the parasitemia infected versus non-infected cells. Ninety four features are found to be statistically significant in discriminating six classes. Here a feature selection-cum-classification scheme has been devised by combining F-statistic, statistical learning techniques i.e., Bayesian learning and support vector machine (SVM) in order to provide the higher classification accuracy using best set of discriminating features. Results show that Bayesian approach provides the highest accuracy i.e., 84% for malaria classification by selecting 19 most significant features while SVM provides highest accuracy i.e., 83.5% with 9 most significant features. Finally, the performance of these two classifiers under feature selection framework has been compared toward malaria parasite classification. Copyright © 2012 Elsevier Ltd. All rights reserved.

Transitional epithelial lesions of the ureter in renal transplant rejection.

PubMed

Katz, J P; Greenstein, S M; Hakki, A; Miller, A; Katz, S M; Simonian, S

1988-04-01

The spectrum of ureteric lesions of human renal allografts, long attributed exclusively to postsurgical complications such as ischemia, has recently been shown to include the types of rejection seen in the kidney. Since the rejected ureter also exhibits transitional epithelial lesions that may impact on renal and ureteral function, we studied, by light, immunohistochemical, immunofluorescent, and electron microscopic techniques, ureters of 65 irreversibly rejected kidneys. Seven unused cadaver kidneys served as controls. Urothelial lesions, noticed in 57 of 65 ureters (88%), ranged from minimal basal vacuolization to complete sloughing with or without necrosis of the epithelial lining. Epithelial exfoliation was noticed in 31 cases (54.4%), and basal vacuolization, severe enough to produce cleavage of the epithelial junctions and thus create bullae, was noticed in 21 cases (36.8%). Immunofluorescent and immunoperoxidase stains, performed in 16 cases, were all positive for immunoglobulins but yielded varied results ranging from granular to linear staining, particularly in the region of the basal cells and the basement membrane. Electron microscopic findings confirmed the light microscopic alterations. By contrast, control ureters showed no lesions. Urothelial ureteric lesions might impede ureteral functions and result in obstruction or infection, thus compounding the consequences of renal allograft rejection. Moreover, elucidation of the pathophysiology of the process will advance the understanding of various cutaneous and transitional epithelial autoimmune conditions.

Visualization of DNA molecules in time during electrophoresis

NASA Technical Reports Server (NTRS)

Lubega, Seth

1991-01-01

For several years individual DNA molecules have been observed and photographed during agarose gel electrophoresis. The DNA molecule is clearly the largest molecule known. Nevertheless, the largest molecule is still too small to be seen using a microscope. A technique developed by Morikawa and Yanagida has made it possible to visualize individual DNA molecules. When these long molecules are labeled with appropriate fluorescence dyes and observed under a fluorescence microscope, although it is not possible to directly visualize the local ultrastructure of the molecules, yet because they are long light emitting chains, their microscopic dynamical behavior can be observed. This visualization works in the same principle that enables one to observe a star through a telescope because it emits light against a dark background. The dynamics of individual DNA molecules migrating through agarose matrix during electrophoresis have been described by Smith et al. (1989), Schwartz and Koval (1989), and Bustamante et al. (1990). DNA molecules during agarose gel electrophoresis advance lengthwise thorough the gel in an extended configuration. They display an extension-contraction motion and tend to bunch up in their leading ends as the 'heads' find new pores through the gel. From time to time they get hooked on obstacles in the gel to form U-shaped configurations before they resume their linear configuration.

Real-time restoration of white-light confocal microscope optical sections

PubMed Central

Balasubramanian, Madhusudhanan; Iyengar, S. Sitharama; Beuerman, Roger W.; Reynaud, Juan; Wolenski, Peter

2009-01-01

Confocal microscopes (CM) are routinely used for building 3-D images of microscopic structures. Nonideal imaging conditions in a white-light CM introduce additive noise and blur. The optical section images need to be restored prior to quantitative analysis. We present an adaptive noise filtering technique using Karhunen–Loéve expansion (KLE) by the method of snapshots and a ringing metric to quantify the ringing artifacts introduced in the images restored at various iterations of iterative Lucy–Richardson deconvolution algorithm. The KLE provides a set of basis functions that comprise the optimal linear basis for an ensemble of empirical observations. We show that most of the noise in the scene can be removed by reconstructing the images using the KLE basis vector with the largest eigenvalue. The prefiltering scheme presented is faster and does not require prior knowledge about image noise. Optical sections processed using the KLE prefilter can be restored using a simple inverse restoration algorithm; thus, the methodology is suitable for real-time image restoration applications. The KLE image prefilter outperforms the temporal-average prefilter in restoring CM optical sections. The ringing metric developed uses simple binary morphological operations to quantify the ringing artifacts and confirms with the visual observation of ringing artifacts in the restored images. PMID:20186290

Contributed review: Review of integrated correlative light and electron microscopy.

PubMed

Timmermans, F J; Otto, C

2015-01-01

New developments in the field of microscopy enable to acquire increasing amounts of information from large sample areas and at an increased resolution. Depending on the nature of the technique, the information may reveal morphological, structural, chemical, and still other sample characteristics. In research fields, such as cell biology and materials science, there is an increasing demand to correlate these individual levels of information and in this way to obtain a better understanding of sample preparation and specific sample properties. To address this need, integrated systems were developed that combine nanometer resolution electron microscopes with optical microscopes, which produce chemically or label specific information through spectroscopy. The complementary information from electron microscopy and light microscopy presents an opportunity to investigate a broad range of sample properties in a correlated fashion. An important part of correlating the differences in information lies in bridging the different resolution and image contrast features. The trend to analyse samples using multiple correlated microscopes has resulted in a new research field. Current research is focused, for instance, on (a) the investigation of samples with nanometer scale distribution of inorganic and organic materials, (b) live cell analysis combined with electron microscopy, and (c) in situ spectroscopic and electron microscopy analysis of catalytic materials, but more areas will benefit from integrated correlative microscopy.

Improving high resolution retinal image quality using speckle illumination HiLo imaging

PubMed Central

Zhou, Xiaolin; Bedggood, Phillip; Metha, Andrew

2014-01-01

Retinal image quality from flood illumination adaptive optics (AO) ophthalmoscopes is adversely affected by out-of-focus light scatter due to the lack of confocality. This effect is more pronounced in small eyes, such as that of rodents, because the requisite high optical power confers a large dioptric thickness to the retina. A recently-developed structured illumination microscopy (SIM) technique called HiLo imaging has been shown to reduce the effect of out-of-focus light scatter in flood illumination microscopes and produce pseudo-confocal images with significantly improved image quality. In this work, we adopted the HiLo technique to a flood AO ophthalmoscope and performed AO imaging in both (physical) model and live rat eyes. The improvement in image quality from HiLo imaging is shown both qualitatively and quantitatively by using spatial spectral analysis. PMID:25136486

Improving high resolution retinal image quality using speckle illumination HiLo imaging.

PubMed

Zhou, Xiaolin; Bedggood, Phillip; Metha, Andrew

2014-08-01

Retinal image quality from flood illumination adaptive optics (AO) ophthalmoscopes is adversely affected by out-of-focus light scatter due to the lack of confocality. This effect is more pronounced in small eyes, such as that of rodents, because the requisite high optical power confers a large dioptric thickness to the retina. A recently-developed structured illumination microscopy (SIM) technique called HiLo imaging has been shown to reduce the effect of out-of-focus light scatter in flood illumination microscopes and produce pseudo-confocal images with significantly improved image quality. In this work, we adopted the HiLo technique to a flood AO ophthalmoscope and performed AO imaging in both (physical) model and live rat eyes. The improvement in image quality from HiLo imaging is shown both qualitatively and quantitatively by using spatial spectral analysis.

Microscopic observation of magnetic bacteria in the magnetic field of a rotating permanent magnet.

PubMed

Smid, Pieter; Shcherbakov, Valeriy; Petersen, Nikolai

2015-09-01

Magnetotactic bacteria are ubiquitous and can be found in both freshwater and marine environments. Due to intracellular chains of magnetic single domain particles, they behave like swimming compass needles. In external magnetic fields like the Earth's magnetic field, a torque is acting on the chain. This will cause the bacterium to be rotated and aligned with the external field. The swimming direction of magnetotactic bacteria can be controlled with external magnetic fields, which makes it convenient to study them under a light microscope. Usually, a special set of coils arranged around a light microscope is used to control the swimming magnetotactic bacteria. Here, we present a simple mechanical system with a permanent magnet, which produces a rotating magnetic field of nearly constant amplitude in the focal plane of a light microscope. The device is placed beside the light microscope and easily adaptable to almost any microscope and thus convenient for field experiments. To describe the trajectories qualitatively, a theoretical model of the trajectories is presented. This device can be used to control the swimming direction of magnetotactic bacteria and also for studying their magnetic and hydrodynamic properties.

Scanning Miniature Microscopes without Lenses

NASA Technical Reports Server (NTRS)

Wang, Yu

2009-01-01

The figure schematically depicts some alternative designs of proposed compact, lightweight optoelectronic microscopes that would contain no lenses and would generate magnified video images of specimens. Microscopes of this type were described previously in Miniature Microscope Without Lenses (NPO - 20218), NASA Tech Briefs, Vol. 22, No. 8 (August 1998), page 43 and Reflective Variants of Miniature Microscope Without Lenses (NPO 20610), NASA Tech Briefs, Vol. 26, No. 9 (September 1999), page 6a. To recapitulate: In the design and construction of a microscope of this type, the focusing optics of a conventional microscope are replaced by a combination of a microchannel filter and a charge-coupled-device (CCD) image detector. Elimination of focusing optics reduces the size and weight of the instrument and eliminates the need for the time-consuming focusing operation. The microscopes described in the cited prior articles contained two-dimensional CCDs registered with two-dimensional arrays of microchannels and, as such, were designed to produce full two-dimensional images, without need for scanning. The microscopes of the present proposal would contain one-dimensional (line image) CCDs registered with linear arrays of microchannels. In the operation of such a microscope, one would scan a specimen along a line perpendicular to the array axis (in other words, one would scan in pushbroom fashion). One could then synthesize a full two-dimensional image of the specimen from the line-image data acquired at one-pixel increments of position along the scan. In one of the proposed microscopes, a beam of unpolarized light for illuminating the specimen would enter from the side. This light would be reflected down onto the specimen by a nonpolarizing beam splitter attached to the microchannels at their lower ends. A portion of the light incident on the specimen would be reflected upward, through the beam splitter and along the microchannels, to form an image on the CCD. If the nonpolarizing beam splitter were replaced by a polarizing one, then the specimen would be illuminated by s-polarized light. Upon reflection from the specimen, some of the s-polarized light would become p-polarized. Only the p-polarized light would contribute to the image on the CCD; in other words, the image would contain information on the polarization rotating characteristic of the specimen.

Methods for characterizing plant fibers.

PubMed

Cruthers, Natasha; Carr, Debra; Niven, Brian; Girvan, Elizabeth; Laing, Raechel

2005-08-01

The effectiveness of different microscopy techniques for measuring the dimensions of ultimate fibers from harakeke (Phormium tenax, New Zealand flax) was investigated using a factorial experimental design. Constant variables were geographical location, location of specimens along the leaf, season (winter), individual plant, a fourth leaf from a north-facing fan, age of plant, and cultivars (two). Experimental variables were microscopy techniques and measurement axis. Measurements of width and length of harakeke ultimate fibers depended on the microscopic preparation/technique used as well as the cultivar examined. The best methods were (i) transverse sections of leaf specimens 4 microm thick, embedded in Paraplast and observed using light microscopy, and (ii) nonfixed ultimate fibers observed using scanning electron microscopy. (c) 2005 Wiley-Liss, Inc.

Laser interferometry force-feedback sensor for an interfacial force microscope

DOEpatents

Houston, Jack E.; Smith, William L.

2004-04-13

A scanning force microscope is provided with a force-feedback sensor to increase sensitivity and stability in determining interfacial forces between a probe and a sample. The sensor utilizes an interferometry technique that uses a collimated light beam directed onto a deflecting member, comprising a common plate suspended above capacitor electrodes situated on a substrate forming an interference cavity with a probe on the side of the common plate opposite the side suspended above capacitor electrodes. The probe interacts with the surface of the sample and the intensity of the reflected beam is measured and used to determine the change in displacement of the probe to the sample and to control the probe distance relative to the surface of the sample.

Innervation of the anterior byssal retractor muscle in Mytilus edulis L. II. Ultrastructure of the glio-interstitial cells.

PubMed

Gilloteaux, J

1975-08-27

Studies on the intrinsic innervation of the anterior byssal retractor muscle (ABRM) in Mytilus edulis L. were continued at the ultrastructural level. Electron micrographs show nerve processes ensheathed by glio-interstitial cells running between muscle fibers. The glio-interstitial cells may represent all the types of osmiophilic cells previously described by the light microscopic ZIO technique in the anterior byssal retractor muscle.

A low-cost, manufacturable method for fabricating capillary and optical fiber interconnects for microfluidic devices.

PubMed

Hartmann, Daniel M; Nevill, J Tanner; Pettigrew, Kenneth I; Votaw, Gregory; Kung, Pang-Jen; Crenshaw, Hugh C

2008-04-01

Microfluidic chips require connections to larger macroscopic components, such as light sources, light detectors, and reagent reservoirs. In this article, we present novel methods for integrating capillaries, optical fibers, and wires with the channels of microfluidic chips. The method consists of forming planar interconnect channels in microfluidic chips and inserting capillaries, optical fibers, or wires into these channels. UV light is manually directed onto the ends of the interconnects using a microscope. UV-curable glue is then allowed to wick to the end of the capillaries, fibers, or wires, where it is cured to form rigid, liquid-tight connections. In a variant of this technique, used with light-guiding capillaries and optical fibers, the UV light is directed into the capillaries or fibers, and the UV-glue is cured by the cone of light emerging from the end of each capillary or fiber. This technique is fully self-aligned, greatly improves both the quality and the manufacturability of the interconnects, and has the potential to enable the fabrication of interconnects in a fully automated fashion. Using these methods, including a semi-automated implementation of the second technique, over 10,000 interconnects have been formed in almost 2000 microfluidic chips made of a variety of rigid materials. The resulting interconnects withstand pressures up to at least 800psi, have unswept volumes estimated to be less than 10 femtoliters, and have dead volumes defined only by the length of the capillary.

Very low risk of light-induced retinal damage during Boston keratoprosthesis surgery: a rabbit study.

PubMed

Salvador-Culla, Borja; Behlau, Irmgard; Sayegh, Rony R; Stacy, Rebecca C; Dohlman, Claes H; Delori, François

2014-02-01

The aim of this study was to assess the possibility of light damage to the retina by a surgical microscope during implantation of a Boston Keratoprosthesis (B-KPro) in rabbits. The retinal irradiance from a Zeiss OPMI Lumera S7 operating microscope was measured at the working distance (16.5 cm). Light transmittance through an isolated B-KPro was measured. A B-KPro was implanted into 1 eye of 12 rabbits with the optic covered during the procedure. The operated eyes were then continuously exposed to a fixed light intensity under the microscope for 1 hour. Fluorescein angiography was carried out on days 2 and 9 postsurgery, after which the animals were euthanized. Further, we compared the potential of these retinal exposures to well-accepted light safety guidelines applicable to humans. Light transmittance of B-KPro revealed a blockage of short wavelengths (<390 nm) and of long wavelengths (1660-1750 nm) of light. In addition, the surgical microscope filtered a part of the blue, ultraviolet, and infrared wavelengths. Neither fluorescein angiography nor a histological examination showed any morphological retinal changes in our rabbits. Moreover, the retinal exposures were well below the safety limits. Modern surgical microscopes have filters incorporated in them that block the most damaging wavelengths of light. The B-KPro is made of 100% poly(methyl methacrylate), which makes it in itself a blocker of short wavelengths of light. No damage could be demonstrated in the animal study, and the retinal exposures were well below the safety limits. Together, these results suggest that light exposures during B-KPro surgery present a low risk of photochemical damage to the retina.

Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy

NASA Astrophysics Data System (ADS)

Jünger, Felix; Olshausen, Philipp V.; Rohrbach, Alexander

2016-07-01

Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes.

Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy

PubMed Central

Jünger, Felix; Olshausen, Philipp v.; Rohrbach, Alexander

2016-01-01

Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes. PMID:27465033

Laser-Stimulated Fluorescence in Paleontology

PubMed Central

Kaye, Thomas G.; Falk, Amanda R.; Pittman, Michael; Sereno, Paul C.; Burnham, David A.; Gong, Enpu; Xu, Xing; Wang, Yinan

2015-01-01

Fluorescence using ultraviolet (UV) light has seen increased use as a tool in paleontology over the last decade. Laser-stimulated fluorescence (LSF) is a next generation technique that is emerging as a way to fluoresce paleontological specimens that remain dark under typical UV. A laser’s ability to concentrate very high flux rates both at the macroscopic and microscopic levels results in specimens fluorescing in ways a standard UV bulb cannot induce. Presented here are five paleontological case histories that illustrate the technique across a broad range of specimens and scales. Novel uses such as back-lighting opaque specimens to reveal detail and detection of specimens completely obscured by matrix are highlighted in these examples. The recent cost reductions in medium-power short wavelength lasers and use of standard photographic filters has now made this technique widely accessible to researchers. This technology has the potential to automate multiple aspects of paleontology, including preparation and sorting of microfossils. This represents a highly cost-effective way to address paleontology's preparatory bottleneck. PMID:26016843

Living cell dry mass measurement using quantitative phase imaging with quadriwave lateral shearing interferometry: an accuracy and sensitivity discussion.

PubMed

Aknoun, Sherazade; Savatier, Julien; Bon, Pierre; Galland, Frédéric; Abdeladim, Lamiae; Wattellier, Benoit; Monneret, Serge

2015-01-01

Single-cell dry mass measurement is used in biology to follow cell cycle, to address effects of drugs, or to investigate cell metabolism. Quantitative phase imaging technique with quadriwave lateral shearing interferometry (QWLSI) allows measuring cell dry mass. The technique is very simple to set up, as it is integrated in a camera-like instrument. It simply plugs onto a standard microscope and uses a white light illumination source. Its working principle is first explained, from image acquisition to automated segmentation algorithm and dry mass quantification. Metrology of the whole process, including its sensitivity, repeatability, reliability, sources of error, over different kinds of samples and under different experimental conditions, is developed. We show that there is no influence of magnification or spatial light coherence on dry mass measurement; effect of defocus is more critical but can be calibrated. As a consequence, QWLSI is a well-suited technique for fast, simple, and reliable cell dry mass study, especially for live cells.

Chemical analyses of fossil bone.

PubMed

Zheng, Wenxia; Schweitzer, Mary Higby

2012-01-01

The preservation of microstructures consistent with soft tissues, cells, and other biological components in demineralized fragments of dinosaur bone tens of millions of years old was unexpected, and counter to current hypotheses of tissue, cellular, and molecular degradation. Although the morphological similarity of these tissues to extant counterparts was unmistakable, after at least 80 million years exposed to geochemical influences, morphological similarity is insufficient to support an endogenous source. To test this hypothesis, and to characterize these materials at a molecular level, we applied multiple independent chemical, molecular, and microscopic analyses to identify the presence of original components produced by the extinct organisms. Microscopic techniques included field emission scanning electron microscopy, analytical transmission electron microscopy, transmitted light microscopy (LM), and fluorescence microscopy (FM). The chemical and molecular techniques include enzyme-linked immunosorbant assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, western blot (immunoblot), and attenuated total reflectance infrared spectroscopy. In situ analyses performed directly on tissues included immunohistochemistry and time-of-flight secondary ion mass spectrometry. The details of sample preparation and methodology are described in detail herein.

Helium Ion Beam Microscopy for Copper Grain Identification in BEOL Structures

NASA Astrophysics Data System (ADS)

van den Boom, Ruud J. J.; Parvaneh, Hamed; Voci, Dave; Huynh, Chuong; Stern, Lewis; Dunn, Kathleen A.; Lifshin, Eric

2009-09-01

Grain size determination in advanced metallization structures requires a technique with resolution ˜2 nm, with a high signal-to-noise ratio and high orientation-dependant contrast for unambiguous identification of grain boundaries. Ideally, such a technique would also be capable of high-throughput and rapid time-to-knowledge. The Helium Ion Microscope (HIM) offers one possibility for achieving these aims in a single platform. This article compares the performance of the HIM with Focused Ion Beam, Scanning Electron and Transmission Electron Microscopes, in terms of achievable image resolution and contrast, using plan-view and cross-sectional imaging of electroplated samples. Although the HIM is capable of sub-nanometer beam diameter, the low signal-to-noise ratio in the images necessitates signal averaging, which degrades the measured image resolution to 6-8 nm. Strategies for improving S/N are discussed in light of the trade-off between beam current and probe size, accelerating voltage, and dwell time.

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.

PubMed

Kaufmann, Anna; Mickoleit, Michaela; Weber, Michael; Huisken, Jan

2012-09-01

Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).

Infrared microscope inspection apparatus

DOEpatents

Forman, S.E.; Caunt, J.W.

1985-02-26

Apparatus and system for inspecting infrared transparents, such as an array of photovoltaic modules containing silicon solar cells, includes an infrared microscope, at least three sources of infrared light placed around and having their axes intersect the center of the object field and means for sending the reflected light through the microscope. The apparatus is adapted to be mounted on an X-Y translator positioned adjacent the object surface. 4 figs.

Infrared microscope inspection apparatus

DOEpatents

Forman, Steven E.; Caunt, James W.

1985-02-26

Apparatus and system for inspecting infrared transparents, such as an array of photovoltaic modules containing silicon solar cells, includes an infrared microscope, at least three sources of infrared light placed around and having their axes intersect the center of the object field and means for sending the reflected light through the microscope. The apparatus is adapted to be mounted on an X-Y translator positioned adjacent the object surface.

[Authentication of Trace Material Evidence in Forensic Science Field with Infrared Microscopic Technique].

PubMed

Jiang, Zhi-quan; Hu, Ke-liang

2016-03-01

In the field of forensic science, conventional infrared spectral analysis technique is usually unable to meet the detection requirements, because only very a few trace material evidence with diverse shapes and complex compositions, can be extracted from the crime scene. Infrared microscopic technique is developed based on a combination of Fourier-transform infrared spectroscopic technique and microscopic technique. Infrared microscopic technique has a lot of advantages over conventional infrared spectroscopic technique, such as high detection sensitivity, micro-area analysisand nondestructive examination. It has effectively solved the problem of authentication of trace material evidence in the field of forensic science. Additionally, almost no external interference is introduced during measurements by infrared microscopic technique. It can satisfy the special need that the trace material evidence must be reserved for witness in court. It is illustrated in detail through real case analysis in this experimental center that, infrared microscopic technique has advantages in authentication of trace material evidence in forensic science field. In this paper, the vibration features in infrared spectra of material evidences, including paints, plastics, rubbers, fibers, drugs and toxicants, can be comparatively analyzed by means of infrared microscopic technique, in an attempt to provide powerful spectroscopic evidence for qualitative diagnosis of various criminal and traffic accident cases. The experimental results clearly suggest that infrared microscopic technique has an incomparable advantage and it has become an effective method for authentication of trace material evidence in the field of forensic science.

A hybrid scanning force and light microscope for surface imaging and three-dimensional optical sectioning in differential interference contrast.

PubMed

Stemmer, A

1995-04-01

The design of a scanned-cantilever-type force microscope is presented which is fully integrated into an inverted high-resolution video-enhanced light microscope. This set-up allows us to acquire thin optical sections in differential interference contrast (DIC) or polarization while the force microscope is in place. Such a hybrid microscope provides a unique platform to study how cell surface properties determine, or are affected by, the three-dimensional dynamic organization inside the living cell. The hybrid microscope presented in this paper has proven reliable and versatile for biological applications. It is the only instrument that can image a specimen by force microscopy and high-power DIC without having either to translate the specimen or to remove the force microscope. Adaptation of the design features could greatly enhance the suitability of other force microscopes for biological work.

To boldly glow ... applications of laser scanning confocal microscopy in developmental biology.

PubMed

Paddock, S W

1994-05-01

The laser scanning confocal microscope (LSCM) is now established as an invaluable tool in developmental biology for improved light microscope imaging of fluorescently labelled eggs, embryos and developing tissues. The universal application of the LSCM in biomedical research has stimulated improvements to the microscopes themselves and the synthesis of novel probes for imaging biological structures and physiological processes. Moreover the ability of the LSCM to produce an optical series in perfect register has made computer 3-D reconstruction and analysis of light microscope images a practical option.

Microscopic Imaging and Spectroscopy with Scattered Light

PubMed Central

Boustany, Nada N.; Boppart, Stephen A.; Backman, Vadim

2012-01-01

Optical contrast based on elastic scattering interactions between light and matter can be used to probe cellular structure and dynamics, and image tissue architecture. The quantitative nature and high sensitivity of light scattering signals to subtle alterations in tissue morphology, as well as the ability to visualize unstained tissue in vivo, has recently generated significant interest in optical scatter based biosensing and imaging. Here we review the fundamental methodologies used to acquire and interpret optical scatter data. We report on recent findings in this field and present current advances in optical scatter techniques and computational methods. Cellular and tissue data enabled by current advances in optical scatter spectroscopy and imaging stand to impact a variety of biomedical applications including clinical tissue diagnosis, in vivo imaging, drug discovery and basic cell biology. PMID:20617940

Visible near-infrared light scattering of single silver split-ring structure made by nanosphere lithography.

PubMed

Okamoto, Toshihiro; Fukuta, Tetsuya; Sato, Shuji; Haraguchi, Masanobu; Fukui, Masuo

2011-04-11

We succeeded in making a silver split-ring (SR) structure of approximately 130 nm in diameter on a glass substrate using a nanosphere lithography technique. The light scattering spectrum in visible near-infrared region of a single, isolated SR was measured using a microscope spectroscopy optical system. The electromagnetic field enhancement spectrum and distribution of the SR structure were simulated by the finite-difference time-domain method, and the excitation modes were clarified. The long wavelength peak in the light scattering spectra corresponded to a fundamental LC resonance mode excited by an incident electric field. It was shown that a single SR structure fabricated as abovementioned can operate as a resonator and generate a magnetic dipole. © 2011 Optical Society of America

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.

Preparation of polymeric Janus particles by directional UV-induced reactions.

PubMed

Liu, Lianying; Ren, Mingwei; Yang, Wantai

2009-09-15

Polymeric Janus particles are obtained by UV-induced selective surface grafting polymerizations and coupling reactions, in virtue of the light-absorption of photoreactive materials such as the immobilized photoinitiator and spread photoinitiator solution on the surfaces exposed to UV light and the sheltering of densely arrayed immovable particles from light. Varying the monomers or macromolecules applied in photografting polymerization or coupling reaction, and choosing diverse polymeric particles of various size, bicolor and amphiphilic Janus particles could be successfully achieved. Observations by fluorescence microscope, scanning electron microscope ,and transmission electron microscope confirmed the asymmetrical morphology of the resultant Janus particles.

Every factor helps: Rapid Ptychographic Reconstruction

NASA Astrophysics Data System (ADS)

Nashed, Youssef

2015-03-01

Recent advances in microscopy, specifically higher spatial resolution and data acquisition rates, require faster and more robust phase retrieval reconstruction methods. Ptychography is a phase retrieval technique for reconstructing the complex transmission function of a specimen from a sequence of diffraction patterns in visible light, X-ray, and electron microscopes. As technical advances allow larger fields to be imaged, computational challenges arise for reconstructing the correspondingly larger data volumes. Waiting to postprocess datasets offline results in missed opportunities. Here we present a parallel method for real-time ptychographic phase retrieval. It uses a hybrid parallel strategy to divide the computation between multiple graphics processing units (GPUs). A final specimen reconstruction is then achieved by different techniques to merge sub-dataset results into a single complex phase and amplitude image. Results are shown on a simulated specimen and real datasets from X-ray experiments conducted at a synchrotron light source.

Hyperspectral imaging with laser-scanning sum-frequency generation microscopy

PubMed Central

Hanninen, Adam; Shu, Ming Wai; Potma, Eric O.

2017-01-01

Vibrationally sensitive sum-frequency generation (SFG) microscopy is a chemically selective imaging technique sensitive to non-centrosymmetric molecular arrangements in biological samples. The routine use of SFG microscopy has been hampered by the difficulty of integrating the required mid-infrared excitation light into a conventional, laser-scanning nonlinear optical (NLO) microscope. In this work, we describe minor modifications to a regular laser-scanning microscope to accommodate SFG microscopy as an imaging modality. We achieve vibrationally sensitive SFG imaging of biological samples with sub-μm resolution at image acquisition rates of 1 frame/s, almost two orders of magnitude faster than attained with previous point-scanning SFG microscopes. Using the fast scanning capability, we demonstrate hyperspectral SFG imaging in the CH-stretching vibrational range and point out its use in the study of molecular orientation and arrangement in biologically relevant samples. We also show multimodal imaging by combining SFG microscopy with second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) on the same imaging platfrom. This development underlines that SFG microscopy is a unique modality with a spatial resolution and image acquisition time comparable to that of other NLO imaging techniques, making point-scanning SFG microscopy a valuable member of the NLO imaging family. PMID:28966861

21 CFR 864.3600 - Microscopes and accessories.

Code of Federal Regulations, 2010 CFR

2010-04-01

... enlarge images of specimens, preparations, and cultures for medical purposes. Variations of microscopes... light. (3) Inverted stage microscopes, which permit examination of tissue cultures or other biological...

Biocytin-Derived MRI Contrast Agent for Longitudinal Brain Connectivity Studies

PubMed Central

2011-01-01

To investigate the connectivity of brain networks noninvasively and dynamically, we have developed a new strategy to functionalize neuronal tracers and designed a biocompatible probe that can be visualized in vivo using magnetic resonance imaging (MRI). Furthermore, the multimodal design used allows combined ex vivo studies with microscopic spatial resolution by conventional histochemical techniques. We present data on the functionalization of biocytin, a well-known neuronal tract tracer, and demonstrate the validity of the approach by showing brain networks of cortical connectivity in live rats under MRI, together with the corresponding microscopic details, such as fibers and neuronal morphology under light microscopy. We further demonstrate that the developed molecule is the first MRI-visible probe to preferentially trace retrograde connections. Our study offers a new platform for the development of multimodal molecular imaging tools of broad interest in neuroscience, that capture in vivo the dynamics of large scale neural networks together with their microscopic characteristics, thereby spanning several organizational levels. PMID:22860157

Mie scattering off coated microbubbles

NASA Astrophysics Data System (ADS)

Nelissen, Radboud; Koene, Elmer; Hilgenfeldt, Sascha; Versluis, Michel

2002-11-01

The acoustic behavior of coated microbubbles depends on parameters of the shell coating, which are in turn dependent on bubble size. More intimate knowledge of this size dependence is required for an improved modeling of a distribution of coated microbubbles such as found in ultrasound contrast agents (UCA). Here a setup is designed to simultaneously measure the optical and acoustic response of an ultrasound-driven single bubble contained in a capillary or levitated by the pressure field of a focused transducer. Optical detection is done by Mie scattering through an inverted microscope. Acoustical detection of the single bubble by a receiving transducer is made possible because of the large working distance of the microscope. For Mie scattering investigation of excited bubbles, two regimes can be distinguished, which require different detection techniques: Conventional wide-angle detection through the microscope objective is sufficient for bubbles of radius exceeding 10 mum. For smaller bubbles, two narrow-aperture detectors are used to reconstruct the bubble dynamics from the complex angle-dependence of the scattered light.

A simple approach to spectrally resolved fluorescence and bright field microscopy over select regions of interest

PubMed Central

Dahlberg, Peter D.; Boughter, Christopher T.; Faruk, Nabil F.; Hong, Lu; Koh, Young Hoon; Reyer, Matthew A.; Sherani, Aiman; Hammond, Adam T.

2016-01-01

A standard wide field inverted microscope was converted to a spatially selective spectrally resolved microscope through the addition of a polarizing beam splitter, a pair of polarizers, an amplitude-mode liquid crystal-spatial light modulator, and a USB spectrometer. The instrument is capable of simultaneously imaging and acquiring spectra over user defined regions of interest. The microscope can also be operated in a bright-field mode to acquire absorption spectra of micron scale objects. The utility of the instrument is demonstrated on three different samples. First, the instrument is used to resolve three differently labeled fluorescent beads in vitro. Second, the instrument is used to recover time dependent bleaching dynamics that have distinct spectral changes in the cyanobacteria, Synechococcus leopoliensis UTEX 625. Lastly, the technique is used to acquire the absorption spectra of CH3NH3PbBr3 perovskites and measure differences between nanocrystal films and micron scale crystals. PMID:27910631

Local Order-Disorder Transition Driving by Structural Heterogeneity in a Benzyl Functionalized Ionic Liquid.

PubMed

Faria, Luiz F O; Paschoal, Vitor H; Lima, Thamires A; Ferreira, Fabio F; Freitas, Rafael S; Ribeiro, Mauro C C

2017-10-26

A local order-disorder transition has been disclosed in the thermophysical behavior of the ionic liquid 1-benzyl-3-methylimidazolium dicyanamide, [Bzmim][N(CN) 2 ], and its microscopic nature revealed by spectroscopic techniques. Differential scanning calorimetry and specific heat measurements show a thermal event of small enthalpy variation taking place in the range 250-260 K, which is not due to crystallization or melting. Molecular dynamic simulations and X-ray diffraction measurements have been used to discuss the segregation of domains in the liquid structure of [Bzmim][N(CN) 2 ]. Raman and NMR spectroscopy measurements as a function of temperature indicate that the microscopic origin of the event observed in the calorimetric measurements comes from structural rearrangement involving the benzyl group. The results indicate that the characteristic structural heterogeneity allow for rearrangements within local domains implying the good glass-forming ability for the low viscosity ionic liquid [Bzmim][N(CN) 2 ]. This work sheds light on our understanding of the microscopic origin behind complex thermal behavior of ionic liquids.

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

PubMed Central

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-01-01

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications. PMID:26525841

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope.

PubMed

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-11-03

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

Modeling brain circuitry over a wide range of scales.

PubMed

Fua, Pascal; Knott, Graham W

2015-01-01

If we are ever to unravel the mysteries of brain function at its most fundamental level, we will need a precise understanding of how its component neurons connect to each other. Electron Microscopes (EM) can now provide the nanometer resolution that is needed to image synapses, and therefore connections, while Light Microscopes (LM) see at the micrometer resolution required to model the 3D structure of the dendritic network. Since both the topology and the connection strength are integral parts of the brain's wiring diagram, being able to combine these two modalities is critically important. In fact, these microscopes now routinely produce high-resolution imagery in such large quantities that the bottleneck becomes automated processing and interpretation, which is needed for such data to be exploited to its full potential. In this paper, we briefly review the Computer Vision techniques we have developed at EPFL to address this need. They include delineating dendritic arbors from LM imagery, segmenting organelles from EM, and combining the two into a consistent representation.

Modeling brain circuitry over a wide range of scales

PubMed Central

Fua, Pascal; Knott, Graham W.

2015-01-01

If we are ever to unravel the mysteries of brain function at its most fundamental level, we will need a precise understanding of how its component neurons connect to each other. Electron Microscopes (EM) can now provide the nanometer resolution that is needed to image synapses, and therefore connections, while Light Microscopes (LM) see at the micrometer resolution required to model the 3D structure of the dendritic network. Since both the topology and the connection strength are integral parts of the brain's wiring diagram, being able to combine these two modalities is critically important. In fact, these microscopes now routinely produce high-resolution imagery in such large quantities that the bottleneck becomes automated processing and interpretation, which is needed for such data to be exploited to its full potential. In this paper, we briefly review the Computer Vision techniques we have developed at EPFL to address this need. They include delineating dendritic arbors from LM imagery, segmenting organelles from EM, and combining the two into a consistent representation. PMID:25904852

Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

NASA Astrophysics Data System (ADS)

Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

2015-11-01

Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

Three-dimensional confocal microscopy of the living cornea and ocular lens

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1991-07-01

The three-dimensional reconstruction of the optic zone of the cornea and the ocular crystalline lens has been accomplished using confocal microscopy and volume rendering computer techniques. A laser scanning confocal microscope was used in the reflected light mode to obtain the two-dimensional images from the cornea and the ocular lens of a freshly enucleated rabbit eye. The light source was an argon ion laser with a 488 nm wavelength. The microscope objective was a Leitz X25, NA 0.6 water immersion lens. The 400 micron thick cornea was optically sectioned into 133 three micron sections. The semi-transparent cornea and the in-situ ocular lens was visualized as high resolution, high contrast two-dimensional images. The structures observed in the cornea include: superficial epithelial cells and their nuclei, basal epithelial cells and their 'beaded' cell borders, basal lamina, nerve plexus, nerve fibers, nuclei of stromal keratocytes, and endothelial cells. The structures observed in the in- situ ocular lens include: lens capsule, lens epithelial cells, and individual lens fibers. The three-dimensional data sets of the cornea and the ocular lens were reconstructed in the computer using volume rendering techniques. Stereo pairs were also created of the two- dimensional ocular images for visualization. The stack of two-dimensional images was reconstructed into a three-dimensional object using volume rendering techniques. This demonstration of the three-dimensional visualization of the intact, enucleated eye provides an important step toward quantitative three-dimensional morphometry of the eye. The important aspects of three-dimensional reconstruction are discussed.

Multistage morphological segmentation of bright-field and fluorescent microscopy images

NASA Astrophysics Data System (ADS)

Korzyńska, A.; Iwanowski, M.

2012-06-01

This paper describes the multistage morphological segmentation method (MSMA) for microscopic cell images. The proposed method enables us to study the cell behaviour by using a sequence of two types of microscopic images: bright field images and/or fluorescent images. The proposed method is based on two types of information: the cell texture coming from the bright field images and intensity of light emission, done by fluorescent markers. The method is dedicated to the image sequences segmentation and it is based on mathematical morphology methods supported by other image processing techniques. The method allows for detecting cells in image independently from a degree of their flattening and from presenting structures which produce the texture. It makes use of some synergic information from the fluorescent light emission image as the support information. The MSMA method has been applied to images acquired during the experiments on neural stem cells as well as to artificial images. In order to validate the method, two types of errors have been considered: the error of cell area detection and the error of cell position using artificial images as the "gold standard".

Virtual microscopes in podiatric medical education.

PubMed

Becker, John H

2006-01-01

In many medical schools, microscopes are being replaced as teaching tools by computers with software that emulates the use of a light microscope. This article chronicles the adoption of "virtual microscopes" by a podiatric medical school and presents the results of educational research on the effectiveness of this adoption in a histology course. If the trend toward virtual microscopy in education continues, many 21st-century physicians will not be trained to operate a light microscope. The replacement of old technologies by new is discussed. The fundamental question is whether all podiatric physicians should be trained in the use of a particular tool or only those who are likely to use it in their own practice.

Numerical dispersion compensation for Partial Coherence Interferometry and Optical Coherence Tomography.

PubMed

Fercher, A; Hitzenberger, C; Sticker, M; Zawadzki, R; Karamata, B; Lasser, T

2001-12-03

Dispersive samples introduce a wavelength dependent phase distortion to the probe beam. This leads to a noticeable loss of depth resolution in high resolution OCT using broadband light sources. The standard technique to avoid this consequence is to balance the dispersion of the sample byarrangingadispersive materialinthereference arm. However, the impact of dispersion is depth dependent. A corresponding depth dependent dispersion balancing technique is diffcult to implement. Here we present a numerical dispersion compensation technique for Partial Coherence Interferometry (PCI) and Optical Coherence Tomography (OCT) based on numerical correlation of the depth scan signal with a depth variant kernel. It can be used a posteriori and provides depth dependent dispersion compensation. Examples of dispersion compensated depth scan signals obtained from microscope cover glasses are presented.

Surface wettability of silicon substrates enhanced by laser ablation

NASA Astrophysics Data System (ADS)

Tseng, Shih-Feng; Hsiao, Wen-Tse; Chen, Ming-Fei; Huang, Kuo-Cheng; Hsiao, Sheng-Yi; Lin, Yung-Sheng; Chou, Chang-Pin

2010-11-01

Laser-ablation techniques have been widely applied for removing material from a solid surface using a laser-beam irradiating apparatus. This paper presents a surface-texturing technique to create rough patterns on a silicon substrate using a pulsed Nd:YAG laser system. The different degrees of microstructure and surface roughness were adjusted by the laser fluence and laser pulse duration. A scanning electron microscope (SEM) and a 3D confocal laser-scanning microscope are used to measure the surface micrograph and roughness of the patterns, respectively. The contact angle variations between droplets on the textured surface were measured using an FTA 188 video contact angle analyzer. The results indicate that increasing the values of laser fluence and laser pulse duration pushes more molten slag piled around these patterns to create micro-sized craters and leads to an increase in the crater height and surface roughness. A typical example of a droplet on a laser-textured surface shows that the droplet spreads very quickly and almost disappears within 0.5167 s, compared to a contact angle of 47.9° on an untextured surface. This processing technique can also be applied to fabricating Si solar panels to increase the absorption efficiency of light.

Dual-modality single particle orientation and rotational tracking of intracellular transport of nanocargos.

PubMed

Sun, Wei; Gu, Yan; Wang, Gufeng; Fang, Ning

2012-01-17

The single particle orientation and rotational tracking (SPORT) technique was introduced recently to follow the rotational motion of plasmonic gold nanorod under a differential interference contrast (DIC) microscope. In biological studies, however, cellular activities usually involve a multiplicity of molecules; thus, tracking the motion of a single molecule/object is insufficient. Fluorescence-based techniques have long been used to follow the spatial and temporal distributions of biomolecules of interest thanks to the availability of multiplexing fluorescent probes. To know the type and number of molecules and the timing of their involvement in a biological process under investigation by SPORT, we constructed a dual-modality DIC/fluorescence microscope to simultaneously image fluorescently tagged biomolecules and plasmonic nanoprobes in living cells. With the dual-modality SPORT technique, the microtubule-based intracellular transport can be unambiguously identified while the dynamic orientation of nanometer-sized cargos can be monitored at video rate. Furthermore, the active transport on the microtubule can be easily separated from the diffusion before the nanocargo docks on the microtubule or after it undocks from the microtubule. The potential of dual-modality SPORT is demonstrated for shedding new light on unresolved questions in intracellular transport.

Multi-scale Observation of Biological Interactions of Nanocarriers: from Nano to Macro

PubMed Central

Jin, Su-Eon; Bae, Jin Woo; Hong, Seungpyo

2010-01-01

Microscopic observations have played a key role in recent advancements in nanotechnology-based biomedical sciences. In particular, multi-scale observation is necessary to fully understand the nano-bio interfaces where a large amount of unprecedented phenomena have been reported. This review describes how to address the physicochemical and biological interactions of nanocarriers within the biological environments using microscopic tools. The imaging techniques are categorized based on the size scale of detection. For observation of the nano-scale biological interactions of nanocarriers, we discuss atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). For the micro to macro-scale (in vitro and in vivo) observation, we focus on confocal laser scanning microscopy (CLSM) as well as in vivo imaging systems such as magnetic resonance imaging (MRI), superconducting quantum interference devices (SQUIDs), and IVIS®. Additionally, recently developed combined techniques such as AFM-CLSM, correlative Light and Electron Microscopy (CLEM), and SEM-spectroscopy are also discussed. In this review, we describe how each technique helps elucidate certain physicochemical and biological activities of nanocarriers such as dendrimers, polymers, liposomes, and polymeric/inorganic nanoparticles, thus providing a toolbox for bioengineers, pharmaceutical scientists, biologists, and research clinicians. PMID:20232368

Acute unclassified leukemia: A clinicopathologic study with diagnostic implications of electron microscopy.

PubMed

Youness, E; Trujillo, J M; Ahearn, M J; McCredie, K B; Cork, A

1980-01-01

By rigid cytological and cytochemical criteria, the diagnosis of acute and undifferentiated leukemia was established in 22 patients. According to defined criteria, the leukemic cells could not be classified by conventional light microscopic techniques employed in the study of hematopoietic tissue. Cytochemical studies including peroxidase, periodic acid schiff (PAS) and nonspecific esterase (alpha napthyl butyrate-reacting esterase) stains were done on fresh bone marrow samples, and the percentage of positive leukemia cells for each of these stains was determined on 200 cells. In this series of leukemias, cytochemistry at the light microscope level did not contribute to further classification. Subsequent electron microscopic examination of bone marrow samples from these patients confirmed the immaturity and nuclear/cytoplasmic asynchrony of the leukemic cells. Several in vivo neoplastic markers, such as nuclear blebs, increased nuclear bodies, and cytoplasmic fibrillar bundles could be demonstrated in these cells. Fourteen cases from this series exhibited peroxidase-positive developmental granule formation at the ultrastructural level and were reclassified as acute granulocyte leukemia (AGL). One case was reclassified as lymphoma (poor differentiated type), one case was diagnosed as acute monocytic leukemia (AmonoL), and six cases remained in the undifferentiated category (AUL). Clinical and laboratory features, response to treatment, and survival data were evaluated for these patients. This study demonstrated that electron microscopy is useful in the cytological diagnosis of human leukemia.

Spectral confocal reflection microscopy using a white light source

NASA Astrophysics Data System (ADS)

Booth, M.; Juškaitis, R.; Wilson, T.

2008-08-01

We present a reflection confocal microscope incorporating a white light supercontinuum source and spectral detection. The microscope provides images resolved spatially in three-dimensions, in addition to spectral resolution covering the wavelength range 450-650nm. Images and reflection spectra of artificial and natural specimens are presented, showing features that are not normally revealed in conventional microscopes or confocal microscopes using discrete line lasers. The specimens include thin film structures on semiconductor chips, iridescent structures in Papilio blumei butterfly scales, nacre from abalone shells and opal gemstones. Quantitative size and refractive index measurements of transparent beads are derived from spectral interference bands.

AFM and x-ray studies of buffing and uv light induced alignment of liquid crystals on SE610 polyimide films

NASA Astrophysics Data System (ADS)

Kim, Jae-Hoon; Shi, Yushan; Ha, Kiryong; West, John L.; Kumar, Satyendra

1997-03-01

We have studied the competition between the effects of mechanical buffing of and photo-induced chemical reaction in Nissan SE610 polyimide film on the director orientation of liquid crystals using atomic force microscopy (AFM) and textural study under polarizing miscroscope. It was found that the uv light exposure after buffing significantly alters the degree and the direction of alignment achieved by buffing. Results of our study show that the two techniques can be used to control and fine-tune liquid crystal alignment. A description of the microscopic changes as inferred from AFM and x-ray studies will be presented.

In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes

PubMed Central

1986-01-01

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick- translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling. PMID:3084498

7 CFR 353.9 - Standards for accreditation of non-government facilities to perform laboratory seed health...

Code of Federal Regulations, 2013 CFR

2013-01-01

... seed requires a stereo microscope. Visual examination of tissue requires a compound light microscope... equipment; fluorescent microscopes; plate readers; spectrophotometers; and the appropriate assay materials...

7 CFR 353.9 - Standards for accreditation of non-government facilities to perform laboratory seed health...

Code of Federal Regulations, 2011 CFR

2011-01-01

... seed requires a stereo microscope. Visual examination of tissue requires a compound light microscope... equipment; fluorescent microscopes; plate readers; spectrophotometers; and the appropriate assay materials...

7 CFR 353.9 - Standards for accreditation of non-government facilities to perform laboratory seed health...

Code of Federal Regulations, 2010 CFR

2010-01-01

... seed requires a stereo microscope. Visual examination of tissue requires a compound light microscope... equipment; fluorescent microscopes; plate readers; spectrophotometers; and the appropriate assay materials...

7 CFR 353.9 - Standards for accreditation of non-government facilities to perform laboratory seed health...

Code of Federal Regulations, 2014 CFR

2014-01-01

... seed requires a stereo microscope. Visual examination of tissue requires a compound light microscope... equipment; fluorescent microscopes; plate readers; spectrophotometers; and the appropriate assay materials...

Dynamic light scattering microscopy

NASA Astrophysics Data System (ADS)

Dzakpasu, Rhonda

An optical microscope technique, dynamic light scattering microscopy (DLSM) that images dynamically scattered light fluctuation decay rates is introduced. Using physical optics we show theoretically that within the optical resolution of the microscope, relative motions between scattering centers are sufficient to produce significant phase variations resulting in interference intensity fluctuations in the image plane. The time scale for these intensity fluctuations is predicted. The spatial coherence distance defining the average distance between constructive and destructive interference in the image plane is calculated and compared with the pixel size. We experimentally tested DLSM on polystyrene latex nanospheres and living macrophage cells. In order to record these rapid fluctuations, on a slow progressive scan CCD camera, we used a thin laser line of illumination on the sample such that only a single column of pixels in the CCD camera is illuminated. This allowed the use of the rate of the column-by-column readout transfer process as the acquisition rate of the camera. This manipulation increased the data acquisition rate by at least an order of magnitude in comparison to conventional CCD cameras rates defined by frames/s. Analysis of the observed fluctuations provides information regarding the rates of motion of the scattering centers. These rates, acquired from each position on the sample are used to create a spatial map of the fluctuation decay rates. Our experiments show that with this technique, we are able to achieve a good signal-to-noise ratio and can monitor fast intensity fluctuations, on the order of milliseconds. DLSM appears to provide dynamic information about fast motions within cells at a sub-optical resolution scale and provides a new kind of spatial contrast.

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.

Microscopic theory of light-induced deformation in amorphous side-chain azobenzene polymers.

PubMed

Toshchevikov, V; Saphiannikova, M; Heinrich, G

2009-04-16

We propose a microscopic theory of light-induced deformation of side-chain azobenzene polymers taking into account the internal structure of polymer chains. Our theory is based on the fact that interaction of chromophores with the polarized light leads to the orientation anisotropy of azobenzene macromolecules which is accompanied by the appearance of mechanical stress. It is the first microscopic theory which provides the value of the light-induced stress larger than the yield stress. This result explains a possibility for the inscription of surface relief gratings in glassy side-chain azobenzene polymers. For some chemical architectures, elongation of a sample demonstrates a nonmonotonic behavior with the light intensity and can change its sign (a stretched sample starts to be uniaxially compressed), in agreement with experiments. Using a viscoplastic approach, we show that the irreversible strain of a sample, which remains after the light is switched off, decreases with increasing temperature and can disappear at certain temperature below the glass transition temperature. This theoretical prediction is also confirmed by recent experiments.

Rapid identification of Salmonella serotypes through hyperspectral microscopy with different lighting sources

USDA-ARS?s Scientific Manuscript database

Hyperspectral microscope imaging (HMI) has the potential to classify foodborne pathogenic bacteria at cell level by combining microscope images with a spectrophotometer. In this study, the spectra generated from HMIs of five live Salmonella serovars from two light sources, metal halide (MH) and tun...

Dual-labeling with 5-aminolevulinic acid and fluorescein for fluorescence-guided resection of high-grade gliomas: technical note.

PubMed

Suero Molina, Eric; Wölfer, Johannes; Ewelt, Christian; Ehrhardt, André; Brokinkel, Benjamin; Stummer, Walter

2018-02-01

OBJECTIVE Fluorescence guidance with 5-aminolevulinic acid (5-ALA) helps improve resections of malignant gliomas. However, one limitation is the low intensity of blue light for background illumination. Fluorescein has recently been reintroduced into neurosurgery, and novel microscope systems are available for visualizing this fluorochrome, which highlights all perfused tissues but has limited selectivity for tumor detection. Here, the authors investigate a combination of both fluorochromes: 5-ALA for distinguishing tumor and fluorescein for providing tissue fluorescence of adjacent brain tissue. METHODS The authors evaluated 6 patients who harbored cerebral lesions suggestive of high-grade glioma. Patients received 5-ALA (20 mg/kg) orally 4 hours before induction of anesthesia. Low-dose fluorescein (3 mg/kg intravenous) was injected immediately after anesthesia induction. Pentero microscopes (equipped either with Yellow 560 or Blue 400 filters) were used to visualize fluorescence. To simultaneously visualize both fluorochromes, the Yellow 560 module was combined with external blue light illumination (D-light C System). RESULTS Fluorescein-induced fluorescence created a useful background for protoporphyrin IX (PPIX) fluorescence, which appeared orange to red, surrounded by greenly fluorescent normal brain and edematous tissue. Green brain-tissue fluorescence was helpful in augmenting background. Levels of blue illumination that were too strong obscured PPIX fluorescence. Unspecific extravasation of fluorescein was noted at resection margins, which did not interfere with PPIX fluorescence detection. CONCLUSIONS Dual labeling with both PPIX and fluorescein fluorescence is feasible and gives superior background information during fluorescence-guided resections. The authors believe that this technique carries potential as a next step in fluorescence-guided resections if it is completely integrated into the surgical microscope.

Development of and Clinical Experience with a Simple Device for Performing Intraoperative Fluorescein Fluorescence Cerebral Angiography: Technical Notes.

PubMed

Ichikawa, Tsuyoshi; Suzuki, Kyouichi; Watanabe, Yoichi; Sato, Taku; Sakuma, Jun; Saito, Kiyoshi

2016-01-01

To perform intraoperative fluorescence angiography (FAG) under a microscope without an integrated FAG function with reasonable cost and sufficient quality for evaluation, we made a small and easy to use device for fluorescein FAG (FAG filter). We investigated the practical use of this FAG filter during aneurysm surgery, revascularization surgery, and brain tumor surgery. The FAG filter consists of two types of filters: an excitatory filter and a barrier filter. The excitatory filter excludes all wavelengths except for blue light and the barrier filter passes long waves except for blue light. By adding this FAG filter to a microscope without an integrated FAG function, light from the microscope illuminating the surgical field becomes blue, which is blocked by the barrier filter. We put the FAG filter on the objective lens of the operating microscope correctly and fluorescein sodium was injected intravenously or intra-arterially. Fluorescence (green light) from vessels in the surgical field and the dyed tumor were clearly observed through the microscope and recorded by a memory device. This method was easy and could be performed in a short time (about 10 seconds). Blood flow of small vessels deep in the surgical field could be observed. Blood flow stagnation could be evaluated. However, images from this method were inferior to those obtained by currently commercially available microscopes with an integrated FAG function. In brain tumor surgery, a stained tumor on the brain surface could be observed using this method. FAG could be performed with a microscope without an integrated FAG function easily with only this FAG filter.

Development of and Clinical Experience with a Simple Device for Performing Intraoperative Fluorescein Fluorescence Cerebral Angiography: Technical Notes

PubMed Central

ICHIKAWA, Tsuyoshi; SUZUKI, Kyouichi; WATANABE, Yoichi; SATO, Taku; SAKUMA, Jun; SAITO, Kiyoshi

2016-01-01

To perform intraoperative fluorescence angiography (FAG) under a microscope without an integrated FAG function with reasonable cost and sufficient quality for evaluation, we made a small and easy to use device for fluorescein FAG (FAG filter). We investigated the practical use of this FAG filter during aneurysm surgery, revascularization surgery, and brain tumor surgery. The FAG filter consists of two types of filters: an excitatory filter and a barrier filter. The excitatory filter excludes all wavelengths except for blue light and the barrier filter passes long waves except for blue light. By adding this FAG filter to a microscope without an integrated FAG function, light from the microscope illuminating the surgical field becomes blue, which is blocked by the barrier filter. We put the FAG filter on the objective lens of the operating microscope correctly and fluorescein sodium was injected intravenously or intra-arterially. Fluorescence (green light) from vessels in the surgical field and the dyed tumor were clearly observed through the microscope and recorded by a memory device. This method was easy and could be performed in a short time (about 10 seconds). Blood flow of small vessels deep in the surgical field could be observed. Blood flow stagnation could be evaluated. However, images from this method were inferior to those obtained by currently commercially available microscopes with an integrated FAG function. In brain tumor surgery, a stained tumor on the brain surface could be observed using this method. FAG could be performed with a microscope without an integrated FAG function easily with only this FAG filter. PMID:26597335

Bright field segmentation tomography (BFST) for use as surface identification in stereomicroscopy

NASA Astrophysics Data System (ADS)

Thiesse, Jacqueline R.; Namati, Eman; de Ryk, Jessica; Hoffman, Eric A.; McLennan, Geoffrey

2004-07-01

Stereomicroscopy is an important method for use in image acquisition because it provides a 3D image of an object when other microscopic techniques can only provide the image in 2D. One challenge that is being faced with this type of imaging is determining the top surface of a sample that has otherwise indistinguishable surface and planar characteristics. We have developed a system that creates oblique illumination and in conjunction with image processing, the top surface can be viewed. The BFST consists of the Leica MZ12 stereomicroscope with a unique attached lighting source. The lighting source consists of eight light emitting diodes (LED's) that are separated by 45-degree angles. Each LED in this system illuminates with a 20-degree viewing angle once per cycle with a shadow over the rest of the sample. Subsequently, eight segmented images are taken per cycle. After the images are captured they are stacked through image addition to achieve the full field of view, and the surface is then easily identified. Image processing techniques, such as skeletonization can be used for further enhancement and measurement. With the use of BFST, advances can be made in detecting surface features from metals to tissue samples, such as in the analytical assessment of pulmonary emphysema using the technique of mean linear intercept.

A technique to calibrate spatial light modulator for varying phase response over its spatial regions

NASA Astrophysics Data System (ADS)

Gupta, Deepak K.; Tata, B. V. R.; Ravindran, T. R.

2018-05-01

Holographic Optical Tweezers (HOTs) employ the technique of beam shaping and holography in an optical manipulation system to create a multitude of focal spots for simultaneous trapping and manipulation of sub-microscopic particles. The beam shaping is accomplished by the use of a phase only liquid crystal spatial light modulator (SLM). The efficiency and the uniformity in the generated traps greatly depend on the phase response behavior of SLMs. In addition the SLMs are found to show different phase response over its different spatial regions, due to non-flat structure of SLMs. Also the phase responses are found to vary over different spatial regions due to non-uniform illumination (Gaussian profile of incident laser). There are various techniques to calibrate for the varying phase response by characterizing the phase modulation at various sub-sections. We present a simple and fast technique to calibrate the SLM suffering with spatially varying phase response. We divide the SLM into many sub-sections and optimize the brightness and gamma of each sub-section for maximum diffraction efficiency. This correction is incorporated in the Weighted Gerchberg Saxton (WGS) algorithm for generation of holograms.

Miniature self-contained vacuum compatible electronic imaging microscope

DOEpatents

Naulleau, Patrick P.; Batson, Phillip J.; Denham, Paul E.; Jones, Michael S.

2001-01-01

A vacuum compatible CCD-based microscopic camera with an integrated illuminator. The camera can provide video or still feed from the microscope contained within a vacuum chamber. Activation of an optional integral illuminator can provide light to illuminate the microscope subject. The microscope camera comprises a housing with a objective port, modified objective, beam-splitter, CCD camera, and LED illuminator.

Design considerations of a real-time clinical confocal microscope

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1991-06-01

A real-time clinical confocal light microscope provides the ophthalmologist with a new tool for the observation of the cornea and the ocular lens. In addition, the ciliary body, the iris, and the sclera can be observed. The real-time light microscopic images have high contrast and resolution. The transverse resolution is about one half micron and the range resolution is one micron. The following observations were made with visible light: corneal epithelial cells, wing cells, basal cells, Bowman's membrane, nerve fibers, basal lamina, fibroblast nuclei, Descemet's membrane, endothelial cells. Observation of the in situ ocular lens showed lens capsule, lens epithelium, lens fibrils, the interior of lens fibrils. The applications of the confocal microscope include: eye banking, laser refractive surgery, observation of wound healing, observation of the iris, the sciera, the ciliary body, the ocular lens, and the intraocular lens. Digital image processing can produce three-dimensional reconstructions of the cornea and the ocular lens.

Using machine-learning to optimize phase contrast in a low-cost cellphone microscope

PubMed Central

Wartmann, Rolf; Schadwinkel, Harald; Heintzmann, Rainer

2018-01-01

Cellphones equipped with high-quality cameras and powerful CPUs as well as GPUs are widespread. This opens new prospects to use such existing computational and imaging resources to perform medical diagnosis in developing countries at a very low cost. Many relevant samples, like biological cells or waterborn parasites, are almost fully transparent. As they do not exhibit absorption, but alter the light’s phase only, they are almost invisible in brightfield microscopy. Expensive equipment and procedures for microscopic contrasting or sample staining often are not available. Dedicated illumination approaches, tailored to the sample under investigation help to boost the contrast. This is achieved by a programmable illumination source, which also allows to measure the phase gradient using the differential phase contrast (DPC) [1, 2] or even the quantitative phase using the derived qDPC approach [3]. By applying machine-learning techniques, such as a convolutional neural network (CNN), it is possible to learn a relationship between samples to be examined and its optimal light source shapes, in order to increase e.g. phase contrast, from a given dataset to enable real-time applications. For the experimental setup, we developed a 3D-printed smartphone microscope for less than 100 $ using off-the-shelf components only such as a low-cost video projector. The fully automated system assures true Koehler illumination with an LCD as the condenser aperture and a reversed smartphone lens as the microscope objective. We show that the effect of a varied light source shape, using the pre-trained CNN, does not only improve the phase contrast, but also the impression of an improvement in optical resolution without adding any special optics, as demonstrated by measurements. PMID:29494620

Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging.

PubMed

Schneider, Gerd; Guttmann, Peter; Rehbein, Stefan; Werner, Stephan; Follath, Rolf

2012-02-01

X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ideally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. In this paper, we describe the X-ray optical set-up and the design of the cryo full-field transmission X-ray microscope (TXM) at the electron storage ring BESSY II. Compared to previous TXM set-ups with zone plate condenser monochromator, the new X-ray optical layout employs an undulator source, a spherical grating monochromator and an elliptically shaped glass capillary mirror as condenser. This set-up improves the spectral resolution by an order of magnitude. Furthermore, the partially coherent object illumination improves the contrast transfer of the microscope compared to incoherent conditions. With the new TXM, cells grown on flat support grids can be tilted perpendicular to the optical axis without any geometrical restrictions by the previously required pinhole for the zone plate monochromator close to the sample plane. We also developed an incorporated fluorescence light microscope which permits to record fluorescence, bright field and DIC images of cryogenic cells inside the TXM. For TXM tomography, imaging with multi-keV X-rays is a straightforward approach to increase the depth of focus. Under these conditions phase contrast imaging is necessary. For soft X-rays with shrinking depth of focus towards 10nm spatial resolution, thin optical sections through a thick specimen might be obtained by deconvolution X-ray microscopy. As alternative 3-D X-ray imaging techniques, the confocal cryo-STXM and the dual beam cryo-FIB/STXM with photoelectron detection are proposed. Copyright © 2012 Elsevier Inc. All rights reserved.

Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope

PubMed Central

Klauss, André; König, Marcelle; Hille, Carsten

2015-01-01

By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED) microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as “easy-STED”, achieving lateral resolution < λ/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating. PMID:26091552

Proper alignment of the microscope.

PubMed

Rottenfusser, Rudi

2013-01-01

The light microscope is merely the first element of an imaging system in a research facility. Such a system may include high-speed and/or high-resolution image acquisition capabilities, confocal technologies, and super-resolution methods of various types. Yet more than ever, the proverb "garbage in-garbage out" remains a fact. Image manipulations may be used to conceal a suboptimal microscope setup, but an artifact-free image can only be obtained when the microscope is optimally aligned, both mechanically and optically. Something else is often overlooked in the quest to get the best image out of the microscope: Proper sample preparation! The microscope optics can only do its job when its design criteria are matched to the specimen or vice versa. The specimen itself, the mounting medium, the cover slip, and the type of immersion medium (if applicable) are all part of the total optical makeup. To get the best results out of a microscope, understanding the functions of all of its variable components is important. Only then one knows how to optimize these components for the intended application. Different approaches might be chosen to discuss all of the microscope's components. We decided to follow the light path which starts with the light source and ends at the camera or the eyepieces. To add more transparency to this sequence, the section up to the microscope stage was called the "Illuminating Section", to be followed by the "Imaging Section" which starts with the microscope objective. After understanding the various components, we can start "working with the microscope." To get the best resolution and contrast from the microscope, the practice of "Koehler Illumination" should be understood and followed by every serious microscopist. Step-by-step instructions as well as illustrations of the beam path in an upright and inverted microscope are included in this chapter. A few practical considerations are listed in Section 3. Copyright © 2013 Elsevier Inc. All rights reserved.

Faster and less phototoxic 3D fluorescence microscopy using a versatile compressed sensing scheme

PubMed Central

Woringer, Maxime; Darzacq, Xavier; Zimmer, Christophe

2017-01-01

Three-dimensional fluorescence microscopy based on Nyquist sampling of focal planes faces harsh trade-offs between acquisition time, light exposure, and signal-to-noise. We propose a 3D compressed sensing approach that uses temporal modulation of the excitation intensity during axial stage sweeping and can be adapted to fluorescence microscopes without hardware modification. We describe implementations on a lattice light sheet microscope and an epifluorescence microscope, and show that images of beads and biological samples can be reconstructed with a 5-10 fold reduction of light exposure and acquisition time. Our scheme opens a new door towards faster and less damaging 3D fluorescence microscopy. PMID:28788909

Cellular localization of Na(+), K(+)-ATPase in the mammalian vestibular system

NASA Technical Reports Server (NTRS)

Kerr, T. P.

1984-01-01

Two different, but complementary, procedures for cellular localization of Na+, K+-ATPase in the guinea pig vestibular system were employed. One of these techniques, devised by Stirling, depends upon the well documented ability of the specific inhibitor ouabain to bind selectively to Na+,K+-ATPase, blocking catalytic activity. Microdisected vestibular tissues are incubated with tritium-labelled (3H-) ouabain, and regions with a high concentration of Na+,K+-ATPase are subsequently identified by light microscope autoradiography. A second method, originated by Ernst, detects inorganic phosphate released from an artificial substrate (nitrophenyl phosphate) by catalytic activity of the enzyme. In the presence of strontium ion, phosphate is precipitated near regions of high activity, then converted to a product which may finally be visualized in the electron microscope. This cytochemical enzymatic reaction is inhibited by ouabain.

Cytochemical features common to nucleoli and cytoplasmic nucleoloids of Olea europaea meiocytes: detection of rRNA by in situ hybridization.

PubMed

Alché, J D; Fernández, M C; Rodríguez-García, M I

1994-02-01

We used light and electron microscopic techniques to study the composition of cytoplasmic nucleoloids during meiotic division in Olea europaea. Nucleoloids were found in two clearly distinguishable morphological varieties: one similar in morphology to the nucleolus, and composed mainly of dense fibrillar component, and another surrounded by many ribosome-like particles. Cytochemical and immunocytochemical techniques showed similar reactivities in nucleoloids and the nucleolus: both are ribonucleoproteic in nature, and possess argyrophillic, argentaffinic and highly phosphorylated proteins. Immunohistochemical techniques failed to detect DNA in either structure. In situ hybridization to a 18 S rRNA probe demonstrated the presence of ribosomal transcripts in both the nucleolus and nucleoloids. These similarities in morphology and composition may reflect similar functionalities.

[Development of a new technique to detect the laterality of microscopic hematuria by means of gas cystoscopy].

PubMed

Kamoi, K; Teraski, T; Kojima, M; Uchida, M; Watanabe, H

1996-04-01

We developed a new technique to determine the laterality of microscopic hematuria by means of gas cystoscopy. An originally designed catheter system consisted of two catheters. On the tip of an inner catheter, a urine dipstick for blood was attached, with a cap on the tip of an outer catheter to keep the dipstick dry. In order to react a dipstick with the urine coming out from a ureteral orifice in the bladder, CO2 was insufflated into the bladder through a cystoscope (gas cystoscopy). The laterality of microscopic hematuria was determined in the bladder, based on the color reaction on the dipstick. This technique was performed successfully in 14 (88%) of 16 cases with microscopic hematuria. The laterality of microscopic hematuria was determined to be ipsilateral in 6 patients, which coincided with the side of a urological upper urinary tract disorder. In contract, bilateral microscopic hematuria was confirmed in 8 patients with glomerular disorders. The diagnostic process in patients with microscopic hematuria remains unsolved for urologists and nephrologists. This technique may provide a new approach in diagnosing microscopic hematuria.

Titania modified activated carbon prepared from sugarcane bagasse: adsorption and photocatalytic degradation of methylene blue under visible light irradiation.

PubMed

El-Salamony, R A; Amdeha, E; Ghoneim, S A; Badawy, N A; Salem, K M; Al-Sabagh, A M

2017-12-01

Activated carbon (AC), prepared from sugarcane bagasse waste through a low-temperature chemical carbonization treatment, was used as a support for nano-TiO 2 . TiO 2 supported on AC (xTiO 2 -AC) catalysts (x = 10, 20, 50, and 70 wt.%) were prepared through a mechano-mixing method. The photocatalysts were characterized by Raman, X-ray diffraction analysis, FTIR, S BET , field emission scanning electron microscope, and optical technique. The adsorption and photo-activity of the prepared catalysts (xTiO 2 -AC) were evaluated using methylene blue (MB) dye. The photocatalytic degradation of MB was evaluated under UVC irradiation and visible light. The degradation percentage of the 100 ppm MB at neutral pH using 20TiO 2 -AC reaches 96 and 91 after 180 min under visible light and UV irradiation, respectively. In other words, these catalysts are more active under visible light than under UV light irradiation, opening the possibility of using solar light for this application.

Sample holder for axial rotation of specimens in 3D microscopy.

PubMed

Bruns, T; Schickinger, S; Schneckenburger, H

2015-10-01

In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three-dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three-dimensional specimens is essential. Fluorescence z-projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Computational imaging of sperm locomotion.

PubMed

Daloglu, Mustafa Ugur; Ozcan, Aydogan

2017-08-01

Not only essential for scientific research, but also in the analysis of male fertility and for animal husbandry, sperm tracking and characterization techniques have been greatly benefiting from computational imaging. Digital image sensors, in combination with optical microscopy tools and powerful computers, have enabled the use of advanced detection and tracking algorithms that automatically map sperm trajectories and calculate various motility parameters across large data sets. Computational techniques are driving the field even further, facilitating the development of unconventional sperm imaging and tracking methods that do not rely on standard optical microscopes and objective lenses, which limit the field of view and volume of the semen sample that can be imaged. As an example, a holographic on-chip sperm imaging platform, only composed of a light-emitting diode and an opto-electronic image sensor, has emerged as a high-throughput, low-cost and portable alternative to lens-based traditional sperm imaging and tracking methods. In this approach, the sample is placed very close to the image sensor chip, which captures lensfree holograms generated by the interference of the background illumination with the light scattered from sperm cells. These holographic patterns are then digitally processed to extract both the amplitude and phase information of the spermatozoa, effectively replacing the microscope objective lens with computation. This platform has further enabled high-throughput 3D imaging of spermatozoa with submicron 3D positioning accuracy in large sample volumes, revealing various rare locomotion patterns. We believe that computational chip-scale sperm imaging and 3D tracking techniques will find numerous opportunities in both sperm related research and commercial applications. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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.

Characterization and identification of microorganisms by FT-IR microspectrometry

NASA Astrophysics Data System (ADS)

Ngo-Thi, N. A.; Kirschner, C.; Naumann, D.

2003-12-01

We report on a novel FT-IR approach for microbial characterization/identification based on a light microscope coupled to an infrared spectrometer which offers the possibility to acquire IR-spectra of microcolonies containing only few hundred cells. Microcolony samples suitable for FT-IR microspectroscopic measurements were obtained by a replica technique with a stamping device that transfers spatially accurate cells of microcolonies growing on solid culture plates to a special, IR-transparent or reflecting stamping plate. High quality spectra could be recorded either by applying the transmission/absorbance or the reflectance/absorbance mode of the infrared microscope. Signal to noise ratios higher than 1000 were obtained for microcolonies as small as 40 μm in diameter. Reproducibility levels were established that allowed species and strain identification. The differentiation and classification capacity of the FT-IR microscopic technique was tested for different selected microorganisms. Cluster and factor analysis methods were used to evaluate the complex spectral data. Excellent discrimination between bacteria and yeasts, and at the same time Gram-negative and Gram-positive bacterial strains was obtained. Twenty-two selected strains of different species within the genus Staphylococcus were repetitively measured and could be grouped into correct species cluster. Moreover, the results indicated that the method allows also identifications at the subspecies level. Additionally, the new approach allowed spectral mapping analysis of single colonies which provided spatially resolved characterization of growth heterogeneity within complex microbial populations such as colonies.

Color digital lensless holographic microscopy: laser versus LED illumination.

PubMed

Garcia-Sucerquia, Jorge

2016-08-20

A comparison of the performance of color digital lensless holographic microscopy (CDLHM) as utilized for illumination of RGB lasers or a super-bright white-light LED with a set of spectral filters is presented. As the use of lasers in CDLHM conceals the possibility of having a compact, lightweight, portable, and low cost microscope, and additionally the limited available laser radiation wavelengths limit a real multispectral imaging microscope, here we present the use of super-bright white-light LED and spectral filters for illuminating the sample. The performance of RGB laser-CDLHM and LED-CDLHM is evaluated on imaging a section of the head of a Drosophila melanogaster fly. This comparison shows that there is trade-off between the spatial resolution of the microscope and the light sources utilized, which can be understood with regard to the coherence properties of the illuminating light. Despite the smaller spatial coherence features of LED-CDLHM in comparison with laser-CDLHM, the former shows promise as a portable RGB digital lensless holographic microscope that could be extended to other wavelengths by the use of different spectral filters.

Dynamic Measurement of Low Contact Angles by Optical Microscopy.

PubMed

Campbell, James M; Christenson, Hugo K

2018-05-16

Precise measurement of contact angles is an important challenge in surface science, in the design and characterization of materials and in many crystallization experiments. Here we present a novel technique for measuring the contact angles of droplets between about 2° and 30°, with the lowest experimental uncertainty at the lower end of this range, typically ±0.1°. The lensing effect of a droplet interface produces the appearance of bright circles in low-aperture light, whose diameter is related to the contact angle. The technique requires no specialized equipment beyond an ordinary optical microscope, and may be used to study the dynamic evolution of the contact angle in situ during an experiment.

Analysis of human hair by Raman microspectroscopy

NASA Astrophysics Data System (ADS)

Plascencia-Castro, A. S.; Cordova-Fraga, T.; Piña-Ruiz, A. L.; Hernández-Rayas, A.; Bernal, J. J.

2017-04-01

Raman microspectroscopy is an optical compound identification technique, which is widely used nowadays for different field applications. A crucial part of this technique is the focus given to the sample in the microscope because it depends on which part of the sample it will analyze. In this work, the effects of irradiating a natural hair samples, obtained from women aged 18 to 55, with a monochromatic light of the Raman spectrometer in two different focus is presented. Two different spectra were obtained with a peak in common. Depending on the information wanted, how the sample is focused plays a crucial role, either way the spectra is information-rich and may be used for biomedical applications.

High resolution IVEM tomography of biological specimens

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

Sedat, J.W.; Agard, D.A.

Electron tomography is a powerful tool for elucidating the three-dimensional architecture of large biological complexes and subcellular organelles. The introduction of intermediate voltage electron microscopes further extended the technique by providing the means to examine very large and non-symmetrical subcellular organelles, at resolutions beyond what would be possible using light microscopy. Recent studies using electron tomography on a variety of cellular organelles and assemblies such as centrosomes, kinetochores, and chromatin have clearly demonstrated the power of this technique for obtaining 3D structural information on non-symmetric cell components. When combined with biochemical and molecular observations, these 3D reconstructions have provided significantmore » new insights into biological function.« less

Transmission X-ray microscopy for full-field nano-imaging of biomaterials

PubMed Central

ANDREWS, JOY C; MEIRER, FLORIAN; LIU, YIJIN; MESTER, ZOLTAN; PIANETTA, PIERO

2010-01-01

Imaging of cellular structure and extended tissue in biological materials requires nanometer resolution and good sample penetration, which can be provided by current full-field transmission X-ray microscopic techniques in the soft and hard X-ray regions. The various capabilities of full-field transmission X-ray microscopy (TXM) include 3D tomography, Zernike phase contrast, quantification of absorption, and chemical identification via X-ray fluorescence and X-ray absorption near edge structure (XANES) imaging. These techniques are discussed and compared in light of results from imaging of biological materials including microorganisms, bone and mineralized tissue and plants, with a focus on hard X-ray TXM at ≤ 40 nm resolution. PMID:20734414

Introduction to the virtual special issue on super-resolution imaging techniques

NASA Astrophysics Data System (ADS)

Cao, Liangcai; Liu, Zhengjun

2017-12-01

Until quite recently, the resolution of optical imaging instruments, including telescopes, cameras and microscopes, was considered to be limited by the diffraction of light and by image sensors. In the past few years, many exciting super-resolution approaches have emerged that demonstrate intriguing ways to bypass the classical limit in optics and detectors. More and more research groups are engaged in the study of advanced super-resolution schemes, devices, algorithms, systems, and applications [1-6]. Super-resolution techniques involve new methods in science and engineering of optics [7,8], measurements [9,10], chemistry [11,12] and information [13,14]. Promising applications, particularly in biomedical research and semiconductor industry, have been successfully demonstrated.

Auricular burns associated with operating microscope use during otologic surgery.

PubMed

Latuska, Richard F; Carlson, Matthew L; Neff, Brian A; Driscoll, Colin L; Wanna, George B; Haynes, David S

2014-02-01

To raise awareness of the potential hazard of auricular burns associated with operating microscope use during otologic surgery. Retrospective case series and summary of the Food and Drug Administration's (FDA) Manufacturer and User Facility Device Experience (MAUDE) database of voluntary adverse event reports pertaining to microscope related auricular thermal injuries. All patients who sustained auricular burns while using the operating microscope during otologic surgery at 2 tertiary academic referral centers. Surgical procedure, microscope model, intensity of illumination, length of procedure, focal length, location and severity of burn, and patient outcome. A total of 4 microscope-related auricular thermal injuries were identified from the authors' institutions. Additionally, 82 unique cases of soft tissue burns associated with the use of an operative microscope have been voluntarily reported to the FDA since 2004. A disproportionately large percent (∼ 30%) of these occurred within the field of otology, the majority of which were during tympanoplasty or tympanomastoidectomy procedures at focal length distances of 300 mm or less with xenon light source microscopes. Simultaneous advancements in light delivery technologies and lens optics have continued to improve the efficiency of the operating microscope; however, these improvements also increase the potential for thermal injuries. Although rare, a review of the FDA MAUDE database suggests that microscope-related soft tissue burns occur more frequently in otology than any other surgical specialty. A variety of factors may help explain this finding, including the unique anatomy of the external ear with thin skin and limited underlying adipose tissue. Preventative measures should be taken to decrease the risk of thermal injuries including use of the lowest comfortable light intensity, adjusting the aperture width to match the operative field, frequent wound irrigation, and covering exposed portions of the pinna with a moist surgical sponge.

Structural, electrical and photovoltaic properties of CoS/Si heterojunction prepared by spray pyrolysis

NASA Astrophysics Data System (ADS)

El Radaf, I. M.; Nasr, Mahmoud; Mansour, A. M.

2018-01-01

Au/p-CoS/n-Si/Al heterojunction device was fabricated by spray pyrolysis technique. The structural and morphological features were examined by x-ray diffraction, scanning electron microscope and energy dispersive x-ray analysis. The capacitance-voltage characteristics of the prepared heterojunction were analyzed at room temperature in the dark. The current-voltage characteristics were examined under dark and different incident light intensities 20-100 mW cm-2. The rectification ratio, series resistance, shunt resistance, diode ideality factor and the effective barrier height were determined at dark and illumination conditions. The photovoltaic parameters such as short circuit current density, open circuit voltage, fill factor and power conversion efficiency were calculated at different incident light intensities.

Utility of fluorescence microscopy in embryonic/fetal topographical analysis.

PubMed

Zucker, R M; Elstein, K H; Shuey, D L; Ebron-McCoy, M; Rogers, J M

1995-06-01

For topographical analysis of developing embryos, investigators typically rely on scanning electron microscopy (SEM) to provide the surface detail not attainable with light microscopy. SEM is an expensive and time-consuming technique, however, and the preparation procedure may alter morphology and leave the specimen friable. We report that by using a high-resolution compound epifluorescence microscope with inexpensive low-power objectives and the fluorochrome acridine orange, we were able to obtain surface images of fixed or fresh whole rat embryos and fetal palates of considerably greater topographical detail than those obtained using routine light microscopy. Indeed the resulting high-resolution images afford not only superior qualitative documentation of morphological observations, but the capability for detailed morphometry via digitization and computer-assisted image analysis.

Any Way You Slice It—A Comparison of Confocal Microscopy Techniques

PubMed Central

Jonkman, James

2015-01-01

The confocal fluorescence microscope has become a popular tool for life sciences researchers, primarily because of its ability to remove blur from outside of the focal plane of the image. Several different kinds of confocal microscopes have been developed, each with advantages and disadvantages. This article will cover the grid confocal, classic confocal laser-scanning microscope (CLSM), the resonant scanning-CLSM, and the spinning-disk confocal microscope. The way each microscope technique works, the best applications the technique is suited for, the limitations of the technique, and new developments for each technology will be presented. Researchers who have access to a range of different confocal microscopes (e.g., through a local core facility) should find this paper helpful for choosing the best confocal technology for specific imaging applications. Others with funding to purchase an instrument should find the article helpful in deciding which technology is ideal for their area of research. PMID:25802490

Foveal light exposure is increased at the time of removal of silicone oil with the potential for phototoxicity.

PubMed

Dogramaci, Mahmut; Williams, Katie; Lee, Ed; Williamson, Tom H

2013-01-01

There is sudden and dramatic visual function deterioration in 1-10 % of eyes filled with silicone oil at the time of removal of silicon oil. Transmission of high-energy blue light is increased in eyes filled with silicone oil. We sought to identify if increased foveal light exposure is a potential factor in the pathophysiology of the visual loss at the time of removal of silicone oil. A graphic ray tracing computer program and laboratory models were used to determine the effect of the intraocular silicone oil bubble size on the foveal illuminance at the time of removal of silicone oil under direct microscope light. The graphic ray tracing computer program revealed a range of optical vignetting effects created by different sizes of silicone oil bubble within the vitreous cavity giving rise to an uneven macular illumination. The laboratory model was used to quantify the variation of illuminance at the foveal region with different sizes of silicone oil bubble with in the vitreous cavity at the time of removal of silicon oil under direct microscope light. To substantiate the hypothesis of the light toxicity during removal of silicone oil, The outcome of oil removal procedures performed under direct microscope illumination in compared to those performed under blocked illumination. The computer program showed that the optical vignetting effect at the macula was dependent on the size of the intraocular silicone oil bubble. The laboratory eye model showed that the foveal illuminance followed a bell-shaped curve with 70 % greater illuminance demonstrated at with 50-60 % silicone oil fill. The clinical data identified five eyes with unexplained vision loss out of 114 eyes that had the procedure performed under direct microscope illumination compared to none out of 78 eyes that had the procedure under blocked illumination. Foveal light exposure, and therefore the potential for phototoxicity, is transiently increased at the time of removal of silicone oil. This is due to uneven macular illumination resulting from the optical vignetting effect of different silicone oil bubble sizes. The increase in foveal light exposure may be significant when the procedure is performed under bright operating microscope light on already stressed photoreceptors of an eye filled with silicon oil. We advocate the use of precautions, such as central shadow filter on the operating microscope light source to reduce foveal light exposure and the risk of phototoxicity at the time of removal of silicone oil. The graphic ray tracing computer program used in this study shows promise in eye modeling for future studies.

Evaluating the use of laser radiation in cleaning of copper embroidery threads on archaeological Egyptian textiles

NASA Astrophysics Data System (ADS)

Abdel-Kareem, Omar; Harith, M. A.

2008-07-01

Cleaning of copper embroidery threads on archaeological textiles is still a complicated conservation process, as most textile conservators believe that the advantages of using traditional cleaning techniques are less than their disadvantages. In this study, the uses of laser cleaning method and two modified recipes of wet cleaning methods were evaluated for cleaning of the corroded archaeological Egyptian copper embroidery threads on an archaeological Egyptian textile fabric. Some corroded copper thread samples were cleaned using modified recipes of wet cleaning method; other corroded copper thread samples were cleaned with Q-switched Nd:YAG laser radiation of wavelength 532 nm. All tested metal thread samples before and after cleaning were investigated using a light microscope and a scanning electron microscope with an energy dispersive X-ray analysis unit. Also the laser-induced breakdown spectroscopy (LIBS) technique was used for the elemental analysis of laser-cleaned samples to follow up the laser cleaning procedure. The results show that laser cleaning is the most effective method among all tested methods in the cleaning of corroded copper threads. It can be used safely in removing the corrosion products without any damage to both metal strips and fibrous core. The tested laser cleaning technique has solved the problems caused by other traditional cleaning techniques that are commonly used in the cleaning of metal threads on museum textiles.

State of the art in advanced endoscopic imaging for the detection and evaluation of dysplasia and early cancer of the gastrointestinal tract.

PubMed

Coda, Sergio; Thillainayagam, Andrew V

2014-01-01

Ideally, endoscopists should be able to detect, characterize, and confirm the nature of a lesion at the bedside, minimizing uncertainties and targeting biopsies and resections only where necessary. However, under conventional white-light inspection - at present, the sole established technique available to most of humanity - premalignant conditions and early cancers can frequently escape detection. In recent years, a range of innovative techniques have entered the endoscopic arena due to their ability to enhance the contrast of diseased tissue regions beyond what is inherently possible with standard white-light endoscopy equipment. The aim of this review is to provide an overview of the state-of-the-art advanced endoscopic imaging techniques available for clinical use that are impacting the way precancerous and neoplastic lesions of the gastrointestinal tract are currently detected and characterized at endoscopy. The basic instrumentation and the physics behind each method, followed by the most influential clinical experience, are described. High-definition endoscopy, with or without optical magnification, has contributed to higher detection rates compared with white-light endoscopy alone and has now replaced ordinary equipment in daily practice. Contrast-enhancement techniques, whether dye-based or computed, have been combined with white-light endoscopy to further improve its accuracy, but histology is still required to clarify the diagnosis. Optical microscopy techniques such as confocal laser endomicroscopy and endocytoscopy enable in vivo histology during endoscopy; however, although of invaluable assistance for tissue characterization, they have not yet made transition between research and clinical use. It is still unknown which approach or combination of techniques offers the best potential. The optimal method will entail the ability to survey wide areas of tissue in concert with the ability to obtain the degree of detailed information provided by microscopic techniques. In this respect, the challenging combination of autofluorescence imaging and confocal endomicroscopy seems promising, and further research is awaited.

In vivo study of rat cortical hemodynamics using a stereotaxic-apparatus-compatible photoacoustic microscope.

PubMed

Guo, Heng; Chen, Qian; Qi, Weizhi; Chen, Xingxing; Xi, Lei

2018-04-19

Brain imaging is an important technique in cognitive neuroscience. In this article, we designed a stereotaxic-apparatus-compatible photoacoustic microscope for the studies of rat cortical hemodynamics. Compared with existing optical resolution photoacoustic microscopy (ORPAM) systems, the probe owns feature of fast, light and miniature. In this microscope, we integrated a miniaturized ultrasound transducer with a center frequency of 10 MHz to detect photoacoustic signals and a 2-dimensional (2D) microelectromechanical system (MEMS) scanner to achieve raster scanning of the optical focus. Based on phantom evaluation, this imaging probe has a high lateral resolution of 3.8 μm and an effective imaging domain of 2 × 2 mm 2 . Different from conventional ORPAMs, combining with standard stereotaxic apparatus enables broad studies of rodent brains without any motion artifact. To show its capability, we successfully captured red blood cell flow in the capillary, monitored the vascular changes during bleeding and blood infusion and visualized cortical hemodynamics induced by middle cerebral artery occlusion. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

sideSPIM - selective plane illumination based on a conventional inverted microscope.

PubMed

Hedde, Per Niklas; Malacrida, Leonel; Ahrar, Siavash; Siryaporn, Albert; Gratton, Enrico

2017-09-01

Previously described selective plane illumination microscopy techniques typically offset ease of use and sample handling for maximum imaging performance or vice versa . Also, to reduce cost and complexity while maximizing flexibility, it is highly desirable to implement light sheet microscopy such that it can be added to a standard research microscope instead of setting up a dedicated system. We devised a new approach termed sideSPIM that provides uncompromised imaging performance and easy sample handling while, at the same time, offering new applications of plane illumination towards fluidics and high throughput 3D imaging of multiple specimen. Based on an inverted epifluorescence microscope, all of the previous functionality is maintained and modifications to the existing system are kept to a minimum. At the same time, our implementation is able to take full advantage of the speed of the employed sCMOS camera and piezo stage to record data at rates of up to 5 stacks/s. Additionally, sample handling is compatible with established methods and switching magnification to change the field of view from single cells to whole organisms does not require labor intensive adjustments of the system.

Enhanced coupling of light into a turbid medium through microscopic interface engineering

PubMed Central

Thompson, Jonathan V.; Hokr, Brett H.; Kim, Wihan; Ballmann, Charles W.; Applegate, Brian E.; Jo, Javier; Yamilov, Alexey; Cao, Hui; Scully, Marlan O.; Yakovlev, Vladislav V.

2017-01-01

There are many optical detection and sensing methods used today that provide powerful ways to diagnose, characterize, and study materials. For example, the measurement of spontaneous Raman scattering allows for remote detection and identification of chemicals. Many other optical techniques provide unique solutions to learn about biological, chemical, and even structural systems. However, when these systems exist in a highly scattering or turbid medium, the optical scattering effects reduce the effectiveness of these methods. In this article, we demonstrate a method to engineer the geometry of the optical interface of a turbid medium, thereby drastically enhancing the coupling efficiency of light into the material. This enhanced optical coupling means that light incident on the material will penetrate deeper into (and through) the medium. It also means that light thus injected into the material will have an enhanced interaction time with particles contained within the material. These results show that, by using the multiple scattering of light in a turbid medium, enhanced light–matter interaction can be achieved; this has a direct impact on spectroscopic methods such as Raman scattering and fluorescence detection in highly scattering regimes. Furthermore, the enhanced penetration depth achieved by this method will directly impact optical techniques that have previously been limited by the inability to deposit sufficient amounts of optical energy below or through highly scattering layers. PMID:28701381

Fiber optic light collection system for scanning-tunneling-microscope-induced light emission.

PubMed

Watkins, Neil J; Long, James P; Kafafi, Zakya H; Mäkinen, Antti J

2007-05-01

We report a compact light collection scheme suitable for retrofitting a scanning tunneling microscope (STM) for STM-induced light emission experiments. The approach uses a pair of optical fibers with large core diameters and high numerical apertures to maximize light collection efficiency and to moderate the mechanical precision required for alignment. Bench tests indicate that efficiency reduction is almost entirely due to reflective losses at the fiber ends, while losses due to fiber misalignment have virtually been eliminated. Photon-map imaging with nanometer features is demonstrated on a stepped Au(111) surface with signal rates exceeding 10(4) counts/s.

Volumetric Light-field Encryption at the Microscopic Scale

PubMed Central

Li, Haoyu; Guo, Changliang; Muniraj, Inbarasan; Schroeder, Bryce C.; Sheridan, John T.; Jia, Shu

2017-01-01

We report a light-field based method that allows the optical encryption of three-dimensional (3D) volumetric information at the microscopic scale in a single 2D light-field image. The system consists of a microlens array and an array of random phase/amplitude masks. The method utilizes a wave optics model to account for the dominant diffraction effect at this new scale, and the system point-spread function (PSF) serves as the key for encryption and decryption. We successfully developed and demonstrated a deconvolution algorithm to retrieve both spatially multiplexed discrete data and continuous volumetric data from 2D light-field images. Showing that the method is practical for data transmission and storage, we obtained a faithful reconstruction of the 3D volumetric information from a digital copy of the encrypted light-field image. The method represents a new level of optical encryption, paving the way for broad industrial and biomedical applications in processing and securing 3D data at the microscopic scale. PMID:28059149

Volumetric Light-field Encryption at the Microscopic Scale

NASA Astrophysics Data System (ADS)

Li, Haoyu; Guo, Changliang; Muniraj, Inbarasan; Schroeder, Bryce C.; Sheridan, John T.; Jia, Shu

2017-01-01

We report a light-field based method that allows the optical encryption of three-dimensional (3D) volumetric information at the microscopic scale in a single 2D light-field image. The system consists of a microlens array and an array of random phase/amplitude masks. The method utilizes a wave optics model to account for the dominant diffraction effect at this new scale, and the system point-spread function (PSF) serves as the key for encryption and decryption. We successfully developed and demonstrated a deconvolution algorithm to retrieve both spatially multiplexed discrete data and continuous volumetric data from 2D light-field images. Showing that the method is practical for data transmission and storage, we obtained a faithful reconstruction of the 3D volumetric information from a digital copy of the encrypted light-field image. The method represents a new level of optical encryption, paving the way for broad industrial and biomedical applications in processing and securing 3D data at the microscopic scale.

Structure and growth of the mesoscopic surfactant/silica thin films

NASA Astrophysics Data System (ADS)

Zhou, Linbo

1999-10-01

We report the study of the structure and the growth of the mesoscopic surfactant/silica thin films. We use X-ray diffraction coupled with Scanning Electron Microscope (SEM), Atomic Force Microscope (AFM), Transmission Electron Microscope (TEM) and light scattering techniques to study the structure, lattice strain and the drying effect of the thin films as well as the growth kinetics and mechanism. The surfactant/silica materials are synthesized using the supramolecular assemblies of the surfactant molecules to template the condensation of the inorganic species. The subsequent calcination yields the mesoporous silica materials, which have many application properties such as unusual electronic, optical, magnetic and elastic characteristics. The films are grown on mica, graphite and silicon substrates in an acidic CTAC (Cetyltrimethyl Ammonium Chloride)/TEOS (Tetraethyl Orthosilicate) solution and are found to consist of the hexagonally packed tubules. The substrate plays an important role in the epitaxial arrangement of the film. We use the light scattering and cryo TEM to study the micelle morphology and aggregation in the solution and use synchrotron radiation X-ray diffraction to study the growth of the film at the solid/liquid interfaces in-situ. An induction time is found followed by the growth of the film at a nonlinear growth rate. The induction time depends on the ratio of the concentrations of CTAC to TEOS in the high CTAC concentration regime. The growth kinetics and mechanism are elucidated in a context of a growth model. For the technological application, Micromolding in Capillaries (MIMIC) technique and the field guided growth are used to process the patterned mesoscopic surfactant/silica thin films and align the nanotubules into the desired orientation. X-ray diffraction characterization has been performed to study the structure and orientation of the thin films. The combined influence of the electric field and the confinement of the mold allows the synthesis of the surfactant/silica thin films with the controlled orientation.

Light ion irradiation for unfavorable soft tissue sarcoma

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

Linstadt, D.; Castro, J.R.; Phillips, T.L.

1990-09-01

Between 1978 and 1989, 32 patients with unfavorable soft tissue sarcoma underwent light ion (helium, neon) irradiation with curative intent at Lawrence Berkeley Laboratory. The tumors were located in the trunk in 22 patients and head and neck in 10. Macroscopic tumor was present in 22 at the time of irradiation. Two patients had tumors apparently induced by previous therapeutic irradiation. Follow-up times for surviving patients ranged from 4 to 121 months (median 27 months). The overall 3-year actuarial local control rate was 62%; the corresponding survival rate was 50%. The 3-year actuarial control rate for patients irradiated with macroscopicmore » tumors was 48%, while none of the patients with microscopic disease developed local recurrence (100%). The corresponding 3-year actuarial survival rates were 40% (macroscopic) and 78% (microscopic). Patients with retroperitoneal sarcoma did notably well; the local control rate and survival rate were 64% and 62%, respectively. Complications were acceptable; there were no radiation related deaths, while two patients (6%) required operations to correct significant radiation-related injuries. These results appear promising compared to those achieved by low -LET irradiation, and suggest that this technique merits further investigation.« less

Immunogold staining procedure for the localisation of regulatory peptides.

PubMed

Varndell, I M; Tapia, F J; Probert, L; Buchan, A M; Gu, J; De Mey, J; Bloom, S R; Polak, J M

1982-01-01

The use of protein A- and IgG-conjugated colloidal gold staining methods for the immuno-localisation of peptide hormones and neurotransmitters at light- and electron microscope level are described and discussed. Bright-field and dark-ground illumination modes have been used to visualise the gold-labelled antigenic sites at the light microscope level. Immunogold staining procedures at the ultrastructural level using region-specific antisera have been adopted to localise specific molecular forms of peptides including gastrin (G17 and G34), glucagon and pro-glucagon, insulin and pro-insulin, in normal tissue and in tumours of the gastroenteropancreatic system. Similar methods have been used to demonstrate the heterogeneity of p-type nerves in the enteric nervous system. Vasoactive intestinal polypeptide (VIP) has been localised to granular sites (mean +/- S.D. granule diameter = 98 +/- 19 nm) in nerve terminals of the enteric plexuses and in tumour cells of diarrhoeogenic VIP-producing neoplasias (mean +/- S.D. granule diameter = 126 +/- 37 nm) using immunogold procedures applied to ultraviolet-cured ultrathin sections. Co-localisation of amines and peptides in carotid body type I cells and in chromaffin cells of normal adrenal medulla and phaeochromocytomas has also been demonstrated. Advantages of the immunogold procedures over alternative immunocytochemical techniques are discussed.

Estimation of polydispersity in aggregating red blood cells by quantitative ultrasound backscatter analysis.

PubMed

de Monchy, Romain; Rouyer, Julien; Destrempes, François; Chayer, Boris; Cloutier, Guy; Franceschini, Emilie

2018-04-01

Quantitative ultrasound techniques based on the backscatter coefficient (BSC) have been commonly used to characterize red blood cell (RBC) aggregation. Specifically, a scattering model is fitted to measured BSC and estimated parameters can provide a meaningful description of the RBC aggregates' structure (i.e., aggregate size and compactness). In most cases, scattering models assumed monodisperse RBC aggregates. This study proposes the Effective Medium Theory combined with the polydisperse Structure Factor Model (EMTSFM) to incorporate the polydispersity of aggregate size. From the measured BSC, this model allows estimating three structural parameters: the mean radius of the aggregate size distribution, the width of the distribution, and the compactness of the aggregates. Two successive experiments were conducted: a first experiment on blood sheared in a Couette flow device coupled with an ultrasonic probe, and a second experiment, on the same blood sample, sheared in a plane-plane rheometer coupled to a light microscope. Results demonstrated that the polydisperse EMTSFM provided the best fit to the BSC data when compared to the classical monodisperse models for the higher levels of aggregation at hematocrits between 10% and 40%. Fitting the polydisperse model yielded aggregate size distributions that were consistent with direct light microscope observations at low hematocrits.

Motion mechanics of non-adherent giant liposomes with a combined optical and atomic force microscope

NASA Astrophysics Data System (ADS)

Moreno-Flores, Susana; Ortíz, Rocío

2017-11-01

Herein we present an investigation of the motional dynamics of single mesoscopic bodies of biological relevance with an AFM-based macromanipulation tool and an optical microscope. Giant liposomes are prominent case examples as minimal cell models; studying their mechanics provides a means to address the influence of structural components in the mechanical behaviour of living cells. However, they also pose an experimental challenge due to their lightness, fragility, and high mobility. Their entrapment in wells in a fluid of lower density allows their study under conditions of constrained motion, which enables the synchronous measurement of nanoforces with motion tracking. The procedure enables to estimate sliding friction coefficients and masses of vesicles, and sheds light upon the region between the vesicle and the underlying substrate. The present study paves the way for the investigation of motion and deformation mechanics with one combined technique and a single type of experiment traditionally vetoed to objects that can move as well as deform. Such an approach can be directly applied to cells in suspension, adherent cells or cellular 3D-assemblies so as to assess substrate biocompatibility, monitor adhesion, detachment, motility as well as deformability.

Bi-directional transmission of molecular information by photon or electron beams passing in the close vicinity of specific molecules, and its clinical and basic research applications: 1) Diagnosis of humans or animal patients without any direct contact; 2) Light microscopic and electron microscopic localization of neuro-transmitters, heavy metals, Oncogen C-fos (AB2), etc. of intracellular fine structures of normal and abnormal single cells using light or electro-microscopic indirect Bi-Digital O-Ring Test.

PubMed

Omura, Y; Losco, M; Omura, A K; Takeshige, C; Hisamitsu, T; Nakajima, H; Soejima, K; Yamamoto, S; Ishikawa, H; Kagoshima, T

1992-01-01

In 1985, Omura, Y. discovered that, when specific molecules were placed anywhere in the close vicinity of the path of a light beam (laser), their molecular information, as well as information on electrical & magnetic fields, is transmitted bi-directionally along the path of this light beam. Namely, this information is transmitted in the direction the light beam is projected and towards the direction from which the light beam is coming. This finding was applied to the following clinical and basic research: 1) In the past, using indirect Bi-Digital O-Ring Test, human or animal patients were diagnosed through an intermediate third person holding a good electrical conducting probe, the tip of which was touching the part of the patient to be examined. However, in order to diagnose the patient in isolation from a distance, or a dangerous or unmanagable unanesthesized animal, such as a lion or tiger, the author succeeded in making a diagnosis by replacing the metal conducting probe with a soft laser beam which is held by the one hand of the third person whose index finger is placed in close vicinity of the laser beam generated by a battery-powered penlight-type solid state laser generator. Thus, diagnosis within visible distance, without direct patient contact, became a reality. 2) Using a projection light microscope, by giving indirect Bi-Digital O-Ring Test while contacting with a fine electro-conductive probe on the magnified fine structure of normal and abnormal cells, various normal and abnormal intracellular substances were localized through a third person holding a pure reference control substance with the same hand that is holding the probe as an intermediary for the indirect Bi-Digital O-Ring Test. Instead of the photon beam in a light microscope, the author found that, using an electron beam passing through the close vicinity of specific molecules of specimens in an electron microscope, the molecular information is transmitted to the magnified fluorescent screen, and an indirect Bi-Digital O-Ring Test could be performed through a projected penlight-type solid state soft laser beam on the magnified intracellular structure through an observation glass window. Using the magnified fine structure of the cells, by either a light projection microscopic field or electron microscope, in various cancer cells of both humans and animals, Oncogen C-fos (AB2) and mercury were found inside of the nucleus. Integrin alpha 5 beta 1 was found on cell membranes and nuclear cell membranes of cancer cells. Acetylcholine was not found anywhere within cancer cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Large-scale tissue clearing (PACT): Technical evaluation and new perspectives in immunofluorescence, histology, and ultrastructure.

PubMed

Neckel, Peter H; Mattheus, Ulrich; Hirt, Bernhard; Just, Lothar; Mack, Andreas F

2016-09-29

Novel techniques, like CLARITY and PACT, render large tissue specimens transparent and thereby suitable for microscopic analysis. We used these techniques to evaluate their potential in the intestine as an exemplary organ with a complex tissue composition. Immunohistochemistry, light sheet-, and confocal scanning-microscopy enabled us to follow complex three-dimensional structures, like nerve fibers, vessels, and epithelial barriers throughout the entire organ. Moreover, in a systematic electron microscopic study, we analyzed the morphology and preservation of tissue on ultrastructural level during the clearing process. We also connect tissue clearing with classical histology and demonstrate that cleared tissues can be stained with Hematoxylin-Eosin and Heidenhain's Azan stain, suggesting potential use in histopathology. These experiments showed that a neutral pH during the clearing process results in much better preservation of tissue ultrastructure and standard stainability. Volume changes of specimens were monitored and quantified during the course of the protocol. Additionally, we employed the technique to visualize the enteric nervous system and the epithelial barrier in post mortem human gut preparations. Our data show the high potential of tissue clearing throughout different tissue types supporting its usefulness in research and diagnosis, and contribute to the technical discussion of ultrastructural tissue-retention.

Large-scale tissue clearing (PACT): Technical evaluation and new perspectives in immunofluorescence, histology, and ultrastructure

PubMed Central

Neckel, Peter H.; Mattheus, Ulrich; Hirt, Bernhard; Just, Lothar; Mack, Andreas F.

2016-01-01

Novel techniques, like CLARITY and PACT, render large tissue specimens transparent and thereby suitable for microscopic analysis. We used these techniques to evaluate their potential in the intestine as an exemplary organ with a complex tissue composition. Immunohistochemistry, light sheet-, and confocal scanning-microscopy enabled us to follow complex three-dimensional structures, like nerve fibers, vessels, and epithelial barriers throughout the entire organ. Moreover, in a systematic electron microscopic study, we analyzed the morphology and preservation of tissue on ultrastructural level during the clearing process. We also connect tissue clearing with classical histology and demonstrate that cleared tissues can be stained with Hematoxylin-Eosin and Heidenhain’s Azan stain, suggesting potential use in histopathology. These experiments showed that a neutral pH during the clearing process results in much better preservation of tissue ultrastructure and standard stainability. Volume changes of specimens were monitored and quantified during the course of the protocol. Additionally, we employed the technique to visualize the enteric nervous system and the epithelial barrier in post mortem human gut preparations. Our data show the high potential of tissue clearing throughout different tissue types supporting its usefulness in research and diagnosis, and contribute to the technical discussion of ultrastructural tissue-retention. PMID:27680942

Preparation of coenzyme Q10 liposomes using supercritical anti-solvent technique.

PubMed

Xia, Fei; Jin, Heyang; Zhao, Yaping; Guo, Xinqiu

2012-01-01

Coenzyme Q(10) (CoQ(10)) proliposomes were prepared using the supercritical anti-solvent (SAS) technique to encapsulate CoQ(10). The mixture of cholesterol and soya bean phosphatidylcholine (PC) was chosen as wall materials. The effects of operation conditions (temperature, pressure and components) on the recovery of CoQ(10) and the CoQ(10) loading in CoQ(10) proliposomes were studied. At the optimum conditions of pressure of 8.0 MPa, temperature of 35°C, the weight ratio of 1/10 between CoQ(10) and PC, and the weight ratio of 1/3 between cholesterol and PC, the CoQ(10) loading reached 8.92%. CoQ(10) liposomes were obtained by hydrating CoQ(10) proliposomes and the entrapment efficiency of CoQ(10) reached 82.28%. The morphologies of CoQ(10) proliposomes were characterized by scanning electron microscope, and their solid states were characterized by X-ray diffractometer. The structures of CoQ(10) liposomes were characterized by transmission electron microscope. The particle size distribution of CoQ(10) liposomes was determined by dynamic light scattering instrument. The results indicate that CoQ(10) liposomes with particle sizes about 50 nm can be easily obtained from hydrating CoQ(10) proliposomes prepared by SAS technique.

Biological applications of confocal fluorescence polarization microscopy

NASA Astrophysics Data System (ADS)

Bigelow, Chad E.

Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of meta-tetrahydroxypheny1chlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proof-of-principle studies are performed in a model system consisting of fluorescently labeled bovine serum albumin attached to sepharose beads. The action of trypsin and proteinase K on the albumin is monitored to demonstrate validity of the technique. Images of the processing of the albumin in J774 murine macrophages are also presented indicating large intercellular differences in enzyme activity. Future directions for the technique are also presented, including the design of enzyme probes specific for prostate specific antigen based on fluorescently-labeled dendrimers. A technique for enzyme imaging based on extracellular autofluorescence is also proposed.

Differentiating characteristic microstructural features of cancerous tissues using Mueller matrix microscope.

PubMed

Wang, Ye; He, Honghui; Chang, Jintao; Zeng, Nan; Liu, Shaoxiong; Li, Migao; Ma, Hui

2015-12-01

Polarized light imaging can provide rich microstructural information of samples, and has been applied to the detections of various abnormal tissues. In this paper, we report a polarized light microscope based on Mueller matrix imaging by adding the polarization state generator and analyzer (PSG and PSA) to a commercial transmission optical microscope. The maximum errors for the absolute values of Mueller matrix elements are reduced to 0.01 after calibration. This Mueller matrix microscope has been used to examine human cervical and liver cancerous tissues with fibrosis. Images of the transformed Mueller matrix parameters provide quantitative assessment on the characteristic features of the pathological tissues. Contrast mechanism of the experimental results are backed up by Monte Carlo simulations based on the sphere-cylinder birefringence model, which reveal the relationship between the pathological features in the cancerous tissues at the cellular level and the polarization parameters. Both the experimental and simulated data indicate that the microscopic transformed Mueller matrix parameters can distinguish the breaking down of birefringent normal tissues for cervical cancer, or the formation of birefringent surrounding structures accompanying the inflammatory reaction for liver cancer. With its simple structure, fast measurement and high precision, polarized light microscope based on Mueller matrix shows a good diagnosis application prospect. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparison of LED and Conventional Fluorescence Microscopy for Detection of Acid Fast Bacilli in a Low-Incidence Setting

PubMed Central

Minion, Jessica; Pai, Madhukar; Ramsay, Andrew; Menzies, Dick; Greenaway, Christina

2011-01-01

Introduction Light emitting diode fluorescence microscopes have many practical advantages over conventional mercury vapour fluorescence microscopes, which would make them the preferred choice for laboratories in both low- and high-resource settings, provided performance is equivalent. Methods In a nested case-control study, we compared diagnostic accuracy and time required to read slides with the Zeiss PrimoStar iLED, LW Scientific Lumin, and a conventional fluorescence microscope (Leica DMLS). Mycobacterial culture was used as the reference standard, and subgroup analysis by specimen source and organism isolated were performed. Results There was no difference in sensitivity or specificity between the three microscopes, and agreement was high for all comparisons and subgroups. The Lumin and the conventional fluorescence microscope were equivalent with respect to time required to read smears, but the Zeiss iLED was significantly time saving compared to both. Conclusions Light emitting diode microscopy should be considered by all tuberculosis diagnostic laboratories, including those in high income countries, as a replacement for conventional fluorescence microscopes. Our findings provide support to the recent World Health Organization policy recommending that conventional fluorescence microscopy be replaced by light emitting diode microscopy using auramine staining in all settings where fluorescence microscopy is currently used. PMID:21811622

Development of HiLo Microscope and its use in In-Vivo Applications

NASA Astrophysics Data System (ADS)

Patel, Shreyas J.

The functionality of achieving optical sectioning in biomedical research is invaluable as it allows for visualization of a biological sample at different depths while being free of background scattering. Most current microscopy techniques that offer optical sectioning, unfortunately, require complex instrumentation and thus are generally costly. HiLo microscopy, on the other hand, offers the same functionality and advantage at a relatively low cost. Hence, the work described in this thesis involves the design, build, and application of a HiLo microscope. More specifically, a standalone HiLo microscope was built in addition to implementing HiLo microscopy on a standard fluorescence microscope. In HiLo microscopy, optical sectioning is achieved by acquiring two different types of images per focal plane. One image is acquired under uniform illumination and the other is acquired under speckle illumination. These images are processed using an algorithm that extracts in-focus information and removes features and glare that occur as a result of background fluorescence. To show the benefits of the HiLo microscopy, several imaging experiments on various samples were performed under a HiLo microscope and compared against a traditional fluorescence microscope and a confocal microscope, which is considered the gold standard in optical imaging. In-vitro and ex-vivo imaging was performed on a set of pollen grains, and optically cleared mouse brain and heart slices. Each of these experiments showed great reduction in background scattering at different depths under HiLo microscopy. More importantly, HiLo imaging of optically cleared heart slice demonstrated emergence of different vasculature at different depths. Reduction of out-of-focus light increased the spatial resolution and allowed better visualization of capillary vessels. Furthermore, HiLo imaging was tested in an in-vivo model of a rodent dorsal window chamber model. When imaging the same sample under confocal microscope, the results were comparable between the two modalities. Additionally, a method of achieving blood flow maps at different depth using a combination of HiLo and LSI imaging is also discussed. The significance of this combined technique could help categorize blood flow to particular depths; this can help improve outcomes of medical treatments such pulse dye laser and photodynamic therapy treatments.

Nano-scale imaging and spectroscopy of plasmonic systems, thermal near-fields, and phase separation in complex oxides

NASA Astrophysics Data System (ADS)

Jones, Andrew C.

Optical spectroscopy represents a powerful characterization technique with the ability to directly interact with the electronic, spin, and lattice excitations in matter. In addition, through implementation of ultrafast techniques, further insight into the real-time dynamics of elementary interactions can be gained. However, the resolution of far-field microscopy techniques is restricted by the diffraction limit setting a spatial resolution limit in the 100s nm to micron range for visible and IR light, respectively. This resolution is too coarse for the characterization of mesoscopic phenomena in condensed matter physics. The development of experimental techniques with nanoscale resolution and sensitivity to optical fields has been a long standing obstacle to the characterization of condensed matter systems on their natural length scales. This dissertation focuses on the fundamental near-field optical properties of surfaces and nanoscale systems as well as the utilization of nano-optical techniques, specifically apertureless scattering-type Scanning Near-field Optical Microscopy (s-SNOM), to characterize said optical properties with nanometer scale resolution. First, the s-SNOM characterization of the field enhancement associated with the localized surface plasmon resonances on metallic structures is discussed. With their ability to localize light, plasmonic nano-structures are promising candidate systems to serve as molecular sensors and nano-photonic devices; however, it is well known that particle morphology and the plasmon resonance alone do not uniquely reflect the details of the local field distribution. Here, I demonstrate the use interferometric s-SNOM for imaging of the near-fields associated with plasmonic resonances of crystalline triangular silver nano-prisms in the visible spectral range. I subsequently show the extension of the concept of a localized plasmon into the mid-IR spectral range with the characterization of near-fields of silver nano-rods. Strong spatial field variation on lengths scales as short as 20 nm is observed associated with the dipolar and quadrupolar modes of both systems with details sensitively depending on the nanoparticle structure and environment. In light of recent publications predicting distinct spectral characteristics of thermal electromagnetic near-fields, I demonstrate the extension of s-SNOM techniques through the implementation of a heated atomic force microscope (AFM) tip acting as its own intrinsic light source for the characterization of thermal near-fields. Here, I detail the spectrally distinct and orders of magnitude enhanced resonant spectral near-field energy density associated with vibrational, phonon, and phonon-polariton modes. Modeling the thermal light scattering by the AFM, the scattering cross-section for thermal light may be related to the electromagnetic local density of states (EM-LDOS) above a surface. Lastly, the unique capability of s-SNOM techniques to characterize phase separation phenomena in correlated electron systems is discussed. This measurement capability provides new microscopic insight into the underlying mechanisms of the rich phase transition behavior exhibited by these materials. As a specific example, the infrared s-SNOM mapping of the metal-insulator transition and the associated nano-domain formation in individual VO2 micro-crystals subject to substrate stress is presented. Our results have important implications for the interpretation of the investigations of conventional polycrystalline thin films where the mutual interaction of constituent crystallites may affect the nature of phase separation processes.

Thermal-Wave Microscope

NASA Technical Reports Server (NTRS)

Jones, Robert E.; Kramarchuk, Ihor; Williams, Wallace D.; Pouch, John J.; Gilbert, Percy

1989-01-01

Computer-controlled thermal-wave microscope developed to investigate III-V compound semiconductor devices and materials. Is nondestructive technique providing information on subsurface thermal features of solid samples. Furthermore, because this is subsurface technique, three-dimensional imaging also possible. Microscope uses intensity-modulated electron beam of modified scanning electron microscope to generate thermal waves in sample. Acoustic waves generated by thermal waves received by transducer and processed in computer to form images displayed on video display of microscope or recorded on magnetic disk.

Application of confocal surface wave microscope to self-calibrated attenuation coefficient measurement by Goos-Hänchen phase shift modulation.

PubMed

Pechprasarn, Suejit; Chow, Terry W K; Somekh, Michael G

2018-06-04

In this paper, we present a direct method to measure surface wave attenuation arising from both ohmic and coupling losses using our recently developed phase spatial light modulator (phase-SLM) based confocal surface plasmon microscope. The measurement is carried out in the far-field using a phase-SLM to impose an artificial surface wave phase profile in the back focal plane (BFP) of a microscope objective. In other words, we effectively provide an artificially engineered backward surface wave by modulating the Goos Hänchen (GH) phase shift of the surface wave. Such waves with opposing phase and group velocities are well known in acoustics and electromagnetic metamaterials but usually require structured or layered surfaces, here the effective wave is produced externally in the microscope illumination path. Key features of the technique developed here are that it (i) is self-calibrating and (ii) can distinguish between attenuation arising from ohmic loss (k″ Ω ) and coupling (reradiation) loss (k″ c ). This latter feature has not been achieved with existing methods. In addition to providing a unique measurement the measurement occurs of over a localized region of a few microns. The results were then validated against the surface plasmons (SP) dip measurement in the BFP and a theoretical model based on a simplified Green's function.

Transition of a dental histology course from light to virtual microscopy.

PubMed

Weaker, Frank J; Herbert, Damon C

2009-10-01

The transition of the dental histology course at the University of Texas Health Science Center at San Antonio Dental School was completed gradually over a five-year period. A pilot project was initially conducted to study the feasibility of integrating virtual microscopy into a traditional light microscopic lecture and laboratory course. Because of the difficulty of procuring quality calcified and decalcified sections of teeth, slides from the student loan collection in the oral histology block of the course were outsourced for conversion to digital images and placed on DVDs along with a slide viewer. The slide viewer mimicked the light microscope, allowing horizontal and vertical movement and changing of magnification, and, in addition, a feature to capture static images. In a survey, students rated the ease of use of the software, quality of the images, maneuverability of the images, and questions regarding use of the software, effective use of laboratory, and faculty time. Because of the positive support from the students, our entire student loan collection of 153 glass slides was subsequently converted to virtual images and distributed on an Apricorn pocket external hard drive. Students were asked to assess the virtual microscope over a four-year period. As a result of the surveys, light microscopes have been totally eliminated, and microscope exams have been replaced with project slide examinations. In the future, we plan to expand our virtual slides and incorporate computer testing.

Light Microscopy Module Fan Disturbance Characterized Through Microgravity Emissions Laboratory Testing

NASA Technical Reports Server (NTRS)

McNelis, Anne M.; Motil, Susan M.

2003-01-01

A Light Microscopy Module (LMM) is being engineered, designed, and developed at the NASA Glenn Research Center. The LMM is planned as a remotely controllable on-orbit microscope subrack facility, allowing flexible scheduling and control of physical science and biological science experiments within Glenn s Fluids Integrated Rack on the International Space Station. The LMM concept is a modified commercial research imaging light microscope with powerful laser-diagnostic hardware and interfaces, creating a one-of-a-kind, state-of-the-art microscopic research facility. The microscope will house several different objectives, corresponding to magnifications of 10, 40, 50, 63, and 100. Features of the LMM include high-resolution color video microscopy, brightfield, darkfield, phase contrast, differential interference contrast, spectrophotometry, and confocal microscopy combined in a single configuration. Also, laser tweezers are integrated with the diagnostics as a sample manipulation technique. As part of the development phase of the LMM, it was necessary to quantify the microgravity disturbances generated by the control box fan. Isolating the fan was deemed necessary to reduce the fan speed harmonic amplitudes and to eliminate any broadband disturbances across the 60- to 70-Hz and 160- to 170-Hz frequency ranges. The accelerations generated by a control box fan component of the LMM were measured in the Microgravity Emissions Laboratory (MEL). The MEL is a low-frequency measurement system developed to simulate and verify the on-orbit International Space Station (ISS) microgravity environment. The accelerations generated by various operating components of the ISS, if too large, could hinder the science performed onboard by disturbing the microgravity environment. The MEL facility gives customers a test-verified way of measuring their compliance with ISS limitations on vibratory disturbance levels. The facility is unique in that inertial forces in 6 degrees of freedom can be characterized simultaneously for an operating test article. Vibratory disturbance levels are measured for engineering or flight-level hardware following development from component to subassembly through the rack-level configuration. The MEL can measure accelerations as small as 10-7g, the accuracy needed to confirm compliance with ISS requirements.

Localization of mitochondria in living cells with rhodamine 123.

PubMed Central

Johnson, L V; Walsh, M L; Chen, L B

1980-01-01

The laser dye rhodamine 123 is shown to be a specific probe for the localization of mitochondria in living cells. By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment. Images PMID:6965798

Hard X-ray full field microscopy and magnifying microtomography using compound refractive lenses

NASA Astrophysics Data System (ADS)

Schroer, Christian G.; Günzler, Til Florian; Benner, Boris; Kuhlmann, Marion; Tümmler, Johannes; Lengeler, Bruno; Rau, Christoph; Weitkamp, Timm; Snigirev, Anatoly; Snigireva, Irina

2001-07-01

For hard X-rays, parabolic compound refractive lenses (PCRLs) are genuine imaging devices like glass lenses for visible light. Based on these new lenses, a hard X-ray full field microscope has been constructed that is ideally suited to image the interior of opaque samples with a minimum of sample preparation. As a result of a large depth of field, CRL micrographs are sharp projection images of most samples. To obtain 3D information about a sample, tomographic techniques are combined with magnified imaging.

Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity

PubMed Central

Boudreau, Colton; Wee, Tse-Luen (Erika); Duh, Yan-Rung (Silvia); Couto, Melissa P.; Ardakani, Kimya H.; Brown, Claire M.

2016-01-01

It is important to determine the most effective method of delivering light onto a specimen for minimal light induced damage. Assays are presented to measure photo-bleaching of fluorophores and photo-toxicity to living cells under different illumination conditions. Turning the light off during part of the experimental time reduced photo-bleaching in a manner proportional to the time of light exposure. The rate of photo-bleaching of EGFP was reduced by 9-fold with light pulsing on the micro-second scale. Similarly, in living cells, rapid line scanning resulted in reduced cell stress as measured by mitochondrial potential, rapid cell protrusion and reduced cell retraction. This was achieved on a commercial confocal laser scanning microscope, without any compromise in image quality, by using rapid laser scan settings and line averaging. Therefore this technique can be implemented broadly without any software or hardware upgrades. Researchers can use the rapid line scanning option to immediately improve image quality on fixed samples, reduce photo-bleaching for large high resolution 3D datasets and improve cell health in live cell experiments. The assays developed here can be applied to other microscopy platforms to measure and optimize light delivery for minimal sample damage and photo-toxicity. PMID:27485088

Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity.

PubMed

Boudreau, Colton; Wee, Tse-Luen Erika; Duh, Yan-Rung Silvia; Couto, Melissa P; Ardakani, Kimya H; Brown, Claire M

2016-08-03

It is important to determine the most effective method of delivering light onto a specimen for minimal light induced damage. Assays are presented to measure photo-bleaching of fluorophores and photo-toxicity to living cells under different illumination conditions. Turning the light off during part of the experimental time reduced photo-bleaching in a manner proportional to the time of light exposure. The rate of photo-bleaching of EGFP was reduced by 9-fold with light pulsing on the micro-second scale. Similarly, in living cells, rapid line scanning resulted in reduced cell stress as measured by mitochondrial potential, rapid cell protrusion and reduced cell retraction. This was achieved on a commercial confocal laser scanning microscope, without any compromise in image quality, by using rapid laser scan settings and line averaging. Therefore this technique can be implemented broadly without any software or hardware upgrades. Researchers can use the rapid line scanning option to immediately improve image quality on fixed samples, reduce photo-bleaching for large high resolution 3D datasets and improve cell health in live cell experiments. The assays developed here can be applied to other microscopy platforms to measure and optimize light delivery for minimal sample damage and photo-toxicity.

3D quantitative phase imaging of neural networks using WDT

NASA Astrophysics Data System (ADS)

Kim, Taewoo; Liu, S. C.; Iyer, Raj; Gillette, Martha U.; Popescu, Gabriel

2015-03-01

White-light diffraction tomography (WDT) is a recently developed 3D imaging technique based on a quantitative phase imaging system called spatial light interference microscopy (SLIM). The technique has achieved a sub-micron resolution in all three directions with high sensitivity granted by the low-coherence of a white-light source. Demonstrations of the technique on single cell imaging have been presented previously; however, imaging on any larger sample, including a cluster of cells, has not been demonstrated using the technique. Neurons in an animal body form a highly complex and spatially organized 3D structure, which can be characterized by neuronal networks or circuits. Currently, the most common method of studying the 3D structure of neuron networks is by using a confocal fluorescence microscope, which requires fluorescence tagging with either transient membrane dyes or after fixation of the cells. Therefore, studies on neurons are often limited to samples that are chemically treated and/or dead. WDT presents a solution for imaging live neuron networks with a high spatial and temporal resolution, because it is a 3D imaging method that is label-free and non-invasive. Using this method, a mouse or rat hippocampal neuron culture and a mouse dorsal root ganglion (DRG) neuron culture have been imaged in order to see the extension of processes between the cells in 3D. Furthermore, the tomogram is compared with a confocal fluorescence image in order to investigate the 3D structure at synapses.

High-Throughput Light Sheet Microscopy for the Automated Live Imaging of Larval Zebrafish

NASA Astrophysics Data System (ADS)

Baker, Ryan; Logan, Savannah; Dudley, Christopher; Parthasarathy, Raghuveer

The zebrafish is a model organism with a variety of useful properties; it is small and optically transparent, it reproduces quickly, it is a vertebrate, and there are a large variety of transgenic animals available. Because of these properties, the zebrafish is well suited to study using a variety of optical technologies including light sheet fluorescence microscopy (LSFM), which provides high-resolution three-dimensional imaging over large fields of view. Research progress, however, is often not limited by optical techniques but instead by the number of samples one can examine over the course of an experiment, which in the case of light sheet imaging has so far been severely limited. Here we present an integrated fluidic circuit and microscope which provides rapid, automated imaging of zebrafish using several imaging modes, including LSFM, Hyperspectral Imaging, and Differential Interference Contrast Microscopy. Using this system, we show that we can increase our imaging throughput by a factor of 10 compared to previous techniques. We also show preliminary results visualizing zebrafish immune response, which is sensitive to gut microbiota composition, and which shows a strong variability between individuals that highlights the utility of high throughput imaging. National Science Foundation, Award No. DBI-1427957.

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.

Electron-Beam-Lithographed Nanostructures as Reference Materials for Label-Free Scattered-Light Biosensing of Single Filoviruses.

PubMed

Agrawal, Anant; Majdi, Joseph; Clouse, Kathleen A; Stantchev, Tzanko

2018-05-23

Optical biosensors based on scattered-light measurements are being developed for rapid and label-free detection of single virions captured from body fluids. Highly controlled, stable, and non-biohazardous reference materials producing virus-like signals are valuable tools to calibrate, evaluate, and refine the performance of these new optical biosensing methods. To date, spherical polymer nanoparticles have been the only non-biological reference materials employed with scattered-light biosensing techniques. However, pathogens like filoviruses, including the Ebola virus, are far from spherical and their shape strongly affects scattered-light signals. Using electron beam lithography, we fabricated nanostructures resembling individual filamentous virions attached to a biosensing substrate (silicon wafer overlaid with silicon oxide film) and characterized their dimensions with scanning electron and atomic force microscopes. To assess the relevance of these nanostructures, we compared their signals across the visible spectrum to signals recorded from Ebola virus-like particles which exhibit characteristic filamentous morphology. We demonstrate the highly stable nature of our nanostructures and use them to obtain new insights into the relationship between virion dimensions and scattered-light signal.

Myosin conformational states determined by single fluorophore polarization

PubMed Central

Warshaw, David M.; Hayes, Eric; Gaffney, Donald; Lauzon, Anne-Marie; Wu, Junru; Kennedy, Guy; Trybus, Kathleen; Lowey, Susan; Berger, Christopher

1998-01-01

Muscle contraction is powered by the interaction of the molecular motor myosin with actin. With new techniques for single molecule manipulation and fluorescence detection, it is now possible to correlate, within the same molecule and in real time, conformational states and mechanical function of myosin. A spot-confocal microscope, capable of detecting single fluorophore polarization, was developed to measure orientational states in the smooth muscle myosin light chain domain during the process of motion generation. Fluorescently labeled turkey gizzard smooth muscle myosin was prepared by removal of endogenous regulatory light chain and re-addition of the light chain labeled at cysteine-108 with the 6-isomer of iodoacetamidotetramethylrhodamine (6-IATR). Single myosin molecule fluorescence polarization data, obtained in a motility assay, provide direct evidence that the myosin light chain domain adopts at least two orientational states during the cyclic interaction of myosin with actin, a randomly disordered state, most likely associated with myosin whereas weakly bound to actin, and an ordered state in which the light chain domain adopts a finite angular orientation whereas strongly bound after the powerstroke. PMID:9653135

Assessing the Penetrating Abilities of Experimental Preparation with Dental Infiltrant Features Using Optical Microscope: Preliminary Study.

PubMed

Skucha-Nowak, Małgorzata; Machorowska-Pieniążek, Agnieszka; Tanasiewicz, Marta

2016-01-01

The aim of the infiltration technique is to penetrate demineralized enamel with a low viscosity resin. Icon® (DMG) is the first ever and so far the only dental infiltrant. Bacteriostaticity is one of the properties that should be inherent in dental infiltrants, but Icon lacks this feature. The aim of the preliminary study was to properly choose a dye which would allow us to assess the penetrating abilities of our own, experimental preparation with features of a dental infiltrant with bacteriostatic properties and to compare using an optical microscope the depth of infiltration of the designed experimental preparation with the infiltrant available on the market. The preparation is supposed to infiltrate decalcified human enamel and be assessed with an optical microscope. Eosin, neutral fuchsine and methylene blue were added to experimental preparation with dental infiltrant features and to Icon® (DMG) in order to assess the depth of penetration of the experimental solution into the decalcified layers of enamel. The experimental solution mixes well with eosin, neutral fuchsine, and methylene blue. During the preliminary study, the authors concluded that the experimental solution mixes well with methylene blue, neutral fuchsine, and eosin. An addition of eosin to a preparation which infiltrates inner, demineralized enamel layers, facilitates the assessment of such a preparation with an optical microscope. A designed experimental solution with the main ingredients, i.e., 2-hydroxyethyl methacrylate (HEMA) and tetraethylene glycol dimethacrylate (TEGDMA) with a ratio of 75% to 25% penetrates the demineralized (decalcified) inner parts of the enamel and polymerizes when exposed to light. In order to assess the infiltration of the experimental solution into the demineralized enamel layers, it is required to improve the measurement techniques that utilize optical microscopy.

Hard x-ray phase contrastmicroscopy - techniques and applications

NASA Astrophysics Data System (ADS)

Holzner, Christian

In 1918, Einstein provided the first description of the nature of the refractive index for X-rays, showing that phase contrast effects are significant. A century later, most x-ray microscopy and nearly all medical imaging remains based on absorption contrast, even though phase contrast offers orders of magnitude improvements in contrast and reduced radiation exposure at multi-keV x-ray energies. The work presented is concerned with developing practical and quantitative methods of phase contrast for x-ray microscopy. A theoretical framework for imaging in phase contrast is put forward; this is used to obtain quantitative images in a scanning microscope using a segmented detector, and to correct for artifacts in a commercial phase contrast x-ray nano-tomography system. The principle of reciprocity between scanning and full-field microscopes is then used to arrive at a novel solution: Zernike contrast in a scanning microscope. These approaches are compared on a theoretical and experimental basis in direct connection with applications using multi-keV x-ray microscopes at the Advanced Photon Source at Argonne National Laboratory. Phase contrast provides the best means to image mass and ultrastructure of light elements that mainly constitute biological matter, while stimulated x-ray fluorescence provides high sensitivity for studies of the distribution of heavier trace elements, such as metals. These approaches are combined in a complementary way to yield quantitative maps of elemental concentration from 2D images, with elements placed in their ultrastructural context. The combination of x-ray fluorescence and phase contrast poses an ideal match for routine, high resolution tomographic imaging of biological samples in the future. The presented techniques and demonstration experiments will help pave the way for this development.

The optics of microscope image formation.

PubMed

Wolf, David E

2013-01-01

Although geometric optics gives a good understanding of how the microscope works, it fails in one critical area, which is explaining the origin of microscope resolution. To accomplish this, one must consider the microscope from the viewpoint of physical optics. This chapter describes the theory of the microscope-relating resolution to the highest spatial frequency that a microscope can collect. The chapter illustrates how Huygens' principle or construction can be used to explain the propagation of a plane wave. It is shown that this limit increases with increasing numerical aperture (NA). As a corollary to this, resolution increases with decreasing wavelength because of how NA depends on wavelength. The resolution is higher for blue light than red light. Resolution is dependent on contrast, and the higher the contrast, the higher the resolution. This last point relates to issues of signal-to-noise and dynamic range. The use of video and new digital cameras has necessitated redefining classical limits such as those of Rayleigh's criterion. Copyright © 2007 Elsevier Inc. All rights reserved.

Novel snapshot hyperspectral imager for fluorescence imaging

NASA Astrophysics Data System (ADS)

Chandler, Lynn; Chandler, Andrea; Periasamy, Ammasi

2018-02-01

Hyperspectral imaging has emerged as a new technique for the identification and classification of biological tissue1. Benefitting recent developments in sensor technology, the new class of hyperspectral imagers can capture entire hypercubes with single shot operation and it shows great potential for real-time imaging in biomedical sciences. This paper explores the use of a SnapShot imager in fluorescence imaging via microscope for the very first time. Utilizing the latest imaging sensor, the Snapshot imager is both compact and attachable via C-mount to any commercially available light microscope. Using this setup, fluorescence hypercubes of several cells were generated, containing both spatial and spectral information. The fluorescence images were acquired with one shot operation for all the emission range from visible to near infrared (VIS-IR). The paper will present the hypercubes obtained images from example tissues (475-630nm). This study demonstrates the potential of application in cell biology or biomedical applications for real time monitoring.

Vapor-condensation-assisted optical microscopy for ultralong carbon nanotubes and other nanostructures.

PubMed

Wang, Jiangtao; Li, Tianyi; Xia, Bingyu; Jin, Xiang; Wei, Haoming; Wu, Wenyun; Wei, Yang; Wang, Jiaping; Liu, Peng; Zhang, Lina; Li, Qunqing; Fan, Shoushan; Jiang, Kaili

2014-06-11

Here we present a simple yet powerful approach for the imaging of nanostructures under an optical microscope with the help of vapor condensation on their surfaces. Supersaturated water vapor will first form a nanometer-sized water droplet on the condensation nuclei on the surface of nanostructures, and then the water droplet will grow bigger and scatter more light to make the outline of the nanostructure be visible under dark-field optical microscope. This vapor-condensation-assisted (VCA) optical microscopy is applicable to a variety of nanostructures from ultralong carbon nanotubes to functional groups, generating images with contrast coming from the difference in density of the condensation sites, and does not induce any impurities to the specimens. Moreover, this low-cost and efficient technique can be conveniently integrated with other facilities, such as Raman spectroscope and so forth, which will pave the way for widespread applications.

Construction and Application of a Terahertz Scanning Near-Field Microscope for Study of Correlated Electron Materials at Cryogenic Temperatures and Nanometer Length Scales

NASA Astrophysics Data System (ADS)

Stinson, Harry Theodore, III

This dissertation describes the design and construction of the world's first cryogenic apertureless near-field microscope designed for terahertz sources and detectors. I first provide motivation for the creation of this instrument in the context of spectroscopy of correlated electron materials, and background information on the two techniques that the instrument combines, scanning near-field optical microscopy and terahertz time-domain spectroscopy. I then detail key components of the instrument design, including proof-of-principle results obtained at room and cryogenic temperatures. Following this, I discuss an imaging experiment performed with this instrument on vanadium dioxide, an insulator-metal transition material, which sheds new light on the nature of the phase transition and provides support for a new model Hamiltonian for the system. Finally, I discuss a theoretical proposal for the study of cuprate superconductors using this instrument.

Aberration correction in wide-field fluorescence microscopy by segmented-pupil image interferometry.

PubMed

Scrimgeour, Jan; Curtis, Jennifer E

2012-06-18

We present a new technique for the correction of optical aberrations in wide-field fluorescence microscopy. Segmented-Pupil Image Interferometry (SPII) uses a liquid crystal spatial light modulator placed in the microscope's pupil plane to split the wavefront originating from a fluorescent object into an array of individual beams. Distortion of the wavefront arising from either system or sample aberrations results in displacement of the images formed from the individual pupil segments. Analysis of image registration allows for the local tilt in the wavefront at each segment to be corrected with respect to a central reference. A second correction step optimizes the image intensity by adjusting the relative phase of each pupil segment through image interferometry. This ensures that constructive interference between all segments is achieved at the image plane. Improvements in image quality are observed when Segmented-Pupil Image Interferometry is applied to correct aberrations arising from the microscope's optical path.

A high-resolution combined scanning laser and widefield polarizing microscope for imaging at temperatures from 4 K to 300 K.

PubMed

Lange, M; Guénon, S; Lever, F; Kleiner, R; Koelle, D

2017-12-01

Polarized light microscopy, as a contrast-enhancing technique for optically anisotropic materials, is a method well suited for the investigation of a wide variety of effects in solid-state physics, as, for example, birefringence in crystals or the magneto-optical Kerr effect (MOKE). We present a microscopy setup that combines a widefield microscope and a confocal scanning laser microscope with polarization-sensitive detectors. By using a high numerical aperture objective, a spatial resolution of about 240 nm at a wavelength of 405 nm is achieved. The sample is mounted on a 4 He continuous flow cryostat providing a temperature range between 4 K and 300 K, and electromagnets are used to apply magnetic fields of up to 800 mT with variable in-plane orientation and 20 mT with out-of-plane orientation. Typical applications of the polarizing microscope are the imaging of the in-plane and out-of-plane magnetization via the longitudinal and polar MOKE, imaging of magnetic flux structures in superconductors covered with a magneto-optical indicator film via the Faraday effect, or imaging of structural features, such as twin-walls in tetragonal SrTiO 3 . The scanning laser microscope furthermore offers the possibility to gain local information on electric transport properties of a sample by detecting the beam-induced voltage change across a current-biased sample. This combination of magnetic, structural, and electric imaging capabilities makes the microscope a viable tool for research in the fields of oxide electronics, spintronics, magnetism, and superconductivity.

A high-resolution combined scanning laser and widefield polarizing microscope for imaging at temperatures from 4 K to 300 K

NASA Astrophysics Data System (ADS)

Lange, M.; Guénon, S.; Lever, F.; Kleiner, R.; Koelle, D.

2017-12-01

Polarized light microscopy, as a contrast-enhancing technique for optically anisotropic materials, is a method well suited for the investigation of a wide variety of effects in solid-state physics, as, for example, birefringence in crystals or the magneto-optical Kerr effect (MOKE). We present a microscopy setup that combines a widefield microscope and a confocal scanning laser microscope with polarization-sensitive detectors. By using a high numerical aperture objective, a spatial resolution of about 240 nm at a wavelength of 405 nm is achieved. The sample is mounted on a 4He continuous flow cryostat providing a temperature range between 4 K and 300 K, and electromagnets are used to apply magnetic fields of up to 800 mT with variable in-plane orientation and 20 mT with out-of-plane orientation. Typical applications of the polarizing microscope are the imaging of the in-plane and out-of-plane magnetization via the longitudinal and polar MOKE, imaging of magnetic flux structures in superconductors covered with a magneto-optical indicator film via the Faraday effect, or imaging of structural features, such as twin-walls in tetragonal SrTiO3. The scanning laser microscope furthermore offers the possibility to gain local information on electric transport properties of a sample by detecting the beam-induced voltage change across a current-biased sample. This combination of magnetic, structural, and electric imaging capabilities makes the microscope a viable tool for research in the fields of oxide electronics, spintronics, magnetism, and superconductivity.

Non-interferometric quantitative phase imaging of yeast cells

NASA Astrophysics Data System (ADS)

Poola, Praveen K.; Pandiyan, Vimal Prabhu; John, Renu

2015-12-01

Real-time imaging of live cells is quite difficult without the addition of external contrast agents. Various methods for quantitative phase imaging of living cells have been proposed like digital holographic microscopy and diffraction phase microscopy. In this paper, we report theoretical and experimental results of quantitative phase imaging of live yeast cells with nanometric precision using transport of intensity equations (TIE). We demonstrate nanometric depth sensitivity in imaging live yeast cells using this technique. This technique being noninterferometric, does not need any coherent light sources and images can be captured through a regular bright-field microscope. This real-time imaging technique would deliver the depth or 3-D volume information of cells and is highly promising in real-time digital pathology applications, screening of pathogens and staging of diseases like malaria as it does not need any preprocessing of samples.

Epifluorescent direct-write photolithography for microfluidic applications

NASA Astrophysics Data System (ADS)

Higgins, MacCallister; Geiger, Emil J.

2015-01-01

We present a technique for fabricating soft-lithography molds created using an epifluorescent microscope. By focusing the UV light emitted from a Hg arc lamp, we demonstrate the ability to direct-write photoresist features with a minimum resolution of 45 μm. This resolution is satisfactory for many microfluidic applications. A major advantage of this technique is its low cost, both in terms of capital investment and on-going expenditures. Furthermore, by using a motorized stage, we can quickly fabricate a design on demand, eliminating the need, cost, and lead-time required for a photomask. With the addition of an electronic shutter, complicated separate structures can be imaged and utilized to make a wide range of microfluidic devices. We demonstrate this technique using dry-film resist due to its low cost, ease of application, and less stringent safety protocols.

Imaging arrangement and microscope

DOEpatents

Pertsinidis, Alexandros; Chu, Steven

2015-12-15

An embodiment of the present invention is an imaging arrangement that includes imaging optics, a fiducial light source, and a control system. In operation, the imaging optics separate light into first and second tight by wavelength and project the first and second light onto first and second areas within first and second detector regions, respectively. The imaging optics separate fiducial light from the fiducial light source into first and second fiducial light and project the first and second fiducial light onto third and fourth areas within the first and second detector regions, respectively. The control system adjusts alignment of the imaging optics so that the first and second fiducial light projected onto the first and second detector regions maintain relatively constant positions within the first and second detector regions, respectively. Another embodiment of the present invention is a microscope that includes the imaging arrangement.

Phototoxic effects of an operating microscope on the ocular surface and tear film.

PubMed

Hwang, Hyung Bin; Kim, Hyun Seung

2014-01-01

We evaluated light exposure-induced dry eye syndrome by investigating the phototoxic effects of an operating microscope on the ocular surface and tear film in rabbits. Sixty eyes of 30 rabbits were divided into 3 groups based on the intensity of light exposure received from an operating microscope: Control group, no exposure to light; group A, 40,000-lx intensity for 30 minutes; and group B, 100,000-lx intensity for 30 minutes. To evaluate the potential damage to the ocular surface and tear film, Schirmer tests, rose bengal staining, and conjunctival impression cytology were performed before the light exposure and at 1, 3, and 5 days afterward. In addition, the expression of interleukin 1-beta was analyzed in tear samples. The expression of mucin 5AC was evaluated using immunofluorescence staining, and periodic acid-Schiff staining was conducted on conjunctival tissues. Corneal and conjunctival tissues were observed by means of electron microscopy. Potential damage to the ocular surface and tear film was found in the light-exposed groups as evidenced by decreased aqueous tear production, devitalized corneal and conjunctival epithelial cells, squamous metaplasia of conjunctival epithelial cells, decreased conjunctival goblet cell density, decreased expression of mucin 5AC, ultrastructural cellular damage to corneal and conjunctival tissues, and increased interleukin 1-beta expression in tears. This damage was more noticeable in group B than in group A (P < 0.05). Light exposure from an operating microscope had phototoxic effects on the ocular surface and tear film in this in vivo experiment. These changes seemed to intensify as the intensity of the light increased. Therefore, excessive light exposure during ophthalmic procedures could be a pathogenic factor in dry eye syndrome after a surgery is performed.

CW laser use in biomedical research and practice

NASA Astrophysics Data System (ADS)

Matthopoulos, D. P.

2003-04-01

The communication of humans with their surrouding is achieved through their senses and the related organs. Visual communication using the eyes is made possible because the various sources of light, natural i.e. the sun or the lightning, or artificial such as Lasers, emit electromagnetic radiation which is either reflected or scattered by surfaces. This radiation received by eyes is processed in the brain where the images of the environment are developed. The luminous processing can be either macro- or microscopic. The macroscopic processing is the result of light coming from the sun or from wide range lamps, while the microscopic results from light coming from wide range lamps, mercury lamps, lasers or electron beam. The microscopic processing is the subject we are dealing with in this presentation.

Study of UV surface plasmons on metallic nanostructures and its applications to nanophotonics

NASA Astrophysics Data System (ADS)

Zhou, Liangcheng

Modern nanotechnology requires the characterization ability in the order of 100 nm or smaller. This resolution requirement cannot be met by using conventional optical microscopy. Nowadays, the mainstream technique that is universally adopted to characterize optical properties on this length scale is Near-field Scanning Optical Microscopy (NSOM). In the effort to improve the resolution and efficiency of NSOM techniques, both nanoscopic fabrication and imaging techniques are critical because the light field strongly intereacts with the metallic NSOM probe or other surfaces to form surface plasmons (SPs). However, much is still unknown about the behavior of light interacting with metallic nanostructures. This calls for research that develops the tool set, methodology and that includes both experimental characterization, and numerical simulations, for the investigation of SPs. The short wavelength of UV light makes it particularly desirable for many industrial processes. So far, little research has been carried out to understand surface plasmon in the UV spectral region. Like conventional optics, UV SPs have unique properties and optical behavior. For this purpose, we modified our existing NSOM into a Photon Scanning Tunneling Microscope (PTSM) and demonstrate its power for the imaging of UV SPs. We present what we believe to be the first direct mapping of the UV SPs on an Al2O3/Al surface. UV SP modes launched by one-dimensional slits or two-dimensional groove arrays and corresponding interference phenomenon were both observed. We then use the same methodology in the engineering of optimized nano aperture such as UV bowtie nanoantenna. For the latter, we find a strong UV intensity profile which is localized to less than 50nm caused by a localized surface plasmon resonance. The relationship of optical field enhancement and antenna geometric shape is studied using numerical simulations and NSOM experiments. In another project, we examine the propagation of light from near-field to far-field. For that purpose, a micro-lens with bull's-eye ring structure, similar to a Fresnel zone plate, is fabricated. We mapped the far-field light distribution from the micro-lens' output by using confocal microscope, which shows that this ring structure exhibit focusing ability as well. Furthermore, we study the ultraviolet (UV) extraordinary optical transmission through nanoslit structures into the far field as well as the localized field enhancement in the near field. The experimental results are compared to numerical modeling results showing good agreement.

Detecting Tie2, an endothelial growth factor receptor, by using immunohistochemistry in mouse lungs.

PubMed

Guha, Prajna P; David, Sascha A; Ghosh, Chandra C

2014-01-01

Immunohistochemical (IHC) staining is an invaluable, sensitive, and effective method to detect the presence and localization of proteins in the cellular compartment in tissues. The basic concept of IHC is detecting the antigen in tissues by means of specific antibody binding, which is then demonstrated with a colored histochemical reaction that can be observed under a light microscope. The most challenging aspect of IHC techniques is optimizing the precise experimental conditions that are required to get a specific and a strong signal. The critical steps of IHC are specimen acquisition, fixation, permeabilization, detection system, and selection of the antigen specific antibody and its optimization. Here, we elaborate the technique using the endothelial growth factor binding receptor Tie2 in mouse lungs.

Single Biomolecules at Cryogenic Temperatures: From Structure to Dynamics

NASA Astrophysics Data System (ADS)

Hofmann, Clemens; Kulzer, Florian; Zondervan, Rob; Köhler, Jürgen; Orrit, Michel

Elucidating the dynamics of proteins remains a central and daunting challenge of molecular biology. In our contribution we discuss the relevance of lowtemperature observations not only to structure, but also to dynamics, and thereby to the function of proteins. We first review investigations on light-harvesting complexes to illustrate how increased photostability at low temperatures and spectral selection provide a deeper insight into the excitonic interactions of the chromophores and the dynamics of the protein scaffold. Furthermore, we introduce a novel technique that achieves controlled, reproducible temperature cycles of a microscopic sample on microsecond timescales. We discuss the potential of this technique as a tool to achieve repeatable single-molecule freeze-trapping and to overcome some of the limitations of single-molecule experiments at room temperature.

Microscopic Shell Model Calculations for sd-Shell Nuclei

NASA Astrophysics Data System (ADS)

Barrett, Bruce R.; Dikmen, Erdal; Maris, Pieter; Shirokov, Andrey M.; Smirnova, Nadya A.; Vary, James P.

Several techniques now exist for performing detailed and accurate calculations of the structure of light nuclei, i.e., A ≤ 16. Going to heavier nuclei requires new techniques or extensions of old ones. One of these is the so-called No Core Shell Model (NCSM) with a Core approach, which involves an Okubo-Lee-Suzuki (OLS) transformation of a converged NCSM result into a single major shell, such as the sd-shell. The obtained effective two-body matrix elements can be separated into core and single-particle (s.p.) energies plus residual two-body interactions, which can be used for performing standard shell-model (SSM) calculations. As an example, an application of this procedure will be given for nuclei at the beginning ofthe sd-shell.

Transmission X-ray microscopy for full-field nano imaging of biomaterials.

PubMed

Andrews, Joy C; Meirer, Florian; Liu, Yijin; Mester, Zoltan; Pianetta, Piero

2011-07-01

Imaging of cellular structure and extended tissue in biological materials requires nanometer resolution and good sample penetration, which can be provided by current full-field transmission X-ray microscopic techniques in the soft and hard X-ray regions. The various capabilities of full-field transmission X-ray microscopy (TXM) include 3D tomography, Zernike phase contrast, quantification of absorption, and chemical identification via X-ray fluorescence and X-ray absorption near edge structure imaging. These techniques are discussed and compared in light of results from the imaging of biological materials including microorganisms, bone and mineralized tissue, and plants, with a focus on hard X-ray TXM at ≤ 40-nm resolution. Copyright © 2010 Wiley-Liss, Inc.

Optofluidic bioimaging platform for quantitative phase imaging of lab on a chip devices using digital holographic microscopy.

PubMed

Pandiyan, Vimal Prabhu; John, Renu

2016-01-20

We propose a versatile 3D phase-imaging microscope platform for real-time imaging of optomicrofluidic devices based on the principle of digital holographic microscopy (DHM). Lab-on-chip microfluidic devices fabricated on transparent polydimethylsiloxane (PDMS) and glass substrates have attained wide popularity in biological sensing applications. However, monitoring, visualization, and characterization of microfluidic devices, microfluidic flows, and the biochemical kinetics happening in these devices is difficult due to the lack of proper techniques for real-time imaging and analysis. The traditional bright-field microscopic techniques fail in imaging applications, as the microfluidic channels and the fluids carrying biological samples are transparent and not visible in bright light. Phase-based microscopy techniques that can image the phase of the microfluidic channel and changes in refractive indices due to the fluids and biological samples present in the channel are ideal for imaging the fluid flow dynamics in a microfluidic channel at high resolutions. This paper demonstrates three-dimensional imaging of a microfluidic device with nanometric depth precisions and high SNR. We demonstrate imaging of microelectrodes of nanometric thickness patterned on glass substrate and the microfluidic channel. Three-dimensional imaging of a transparent PDMS optomicrofluidic channel, fluid flow, and live yeast cell flow in this channel has been demonstrated using DHM. We also quantify the average velocity of fluid flow through the channel. In comparison to any conventional bright-field microscope, the 3D depth information in the images illustrated in this work carry much information about the biological system under observation. The results demonstrated in this paper prove the high potential of DHM in imaging optofluidic devices; detection of pathogens, cells, and bioanalytes on lab-on-chip devices; and in studying microfluidic dynamics in real time based on phase changes.

Development of a versatile laser light scattering instrument

NASA Astrophysics Data System (ADS)

Meyer, William V.; Ansari, Rafat R.

1990-10-01

A versatile laser light scattering (LLS) instrument is developed for use in microgravity to measure microscopic particles of 30 A to above 3 microns. Since it is an optical technique, LLS does not affect the sample being studied. A LLS instrument built from modules allows several configurations, each optimized for a particular experiment. The multiangle LLS instrument can be mounted in the rack in the Space Shuttle and on Space Station Freedom. It is possible that a Space Shuttle glove-box and a lap-top computer containing a correlator card can be used to perform a number of experiments and to demonstrate the technology needed for more elaborate investigations. This offers simple means of flying a great number of experiments without the additional requirements of full-scale flight hardware experiments.

Development of a versatile laser light scattering instrument

NASA Technical Reports Server (NTRS)

Meyer, William V.; Ansari, Rafat R.

1990-01-01

A versatile laser light scattering (LLS) instrument is developed for use in microgravity to measure microscopic particles of 30 A to above 3 microns. Since it is an optical technique, LLS does not affect the sample being studied. A LLS instrument built from modules allows several configurations, each optimized for a particular experiment. The multiangle LLS instrument can be mounted in the rack in the Space Shuttle and on Space Station Freedom. It is possible that a Space Shuttle glove-box and a lap-top computer containing a correlator card can be used to perform a number of experiments and to demonstrate the technology needed for more elaborate investigations. This offers simple means of flying a great number of experiments without the additional requirements of full-scale flight hardware experiments.

Growth and characterization of thin Cu-phthalocyanine films on MgO(001) layer for organic light-emitting diodes

PubMed Central

2012-01-01

Surface morphology and thermal stability of Cu-phthalocyanine (CuPc) films grown on an epitaxially grown MgO(001) layer were investigated by using atomic force microscope and X-ray diffractometer. The (002) textured β phase of CuPc films were prepared at room temperature beyond the epitaxial MgO/Fe/MgO(001) buffer layer by the vacuum deposition technique. The CuPc structure remained stable even after post-annealing at 350°C for 1 h under vacuum, which is an important advantage of device fabrication. In order to improve the device performance, we investigated also current-voltage-luminescence characteristics for the new top-emitting organic light-emitting diodes with different thicknesses of CuPc layer. PMID:23181826

Growth and characterization of thin Cu-phthalocyanine films on MgO(001) layer for organic light-emitting diodes.

PubMed

Bae, Yu Jeong; Lee, Nyun Jong; Kim, Tae Hee; Cho, Hyunduck; Lee, Changhee; Fleet, Luke; Hirohata, Atsufumi

2012-11-26

Surface morphology and thermal stability of Cu-phthalocyanine (CuPc) films grown on an epitaxially grown MgO(001) layer were investigated by using atomic force microscope and X-ray diffractometer. The (002) textured β phase of CuPc films were prepared at room temperature beyond the epitaxial MgO/Fe/MgO(001) buffer layer by the vacuum deposition technique. The CuPc structure remained stable even after post-annealing at 350°C for 1 h under vacuum, which is an important advantage of device fabrication. In order to improve the device performance, we investigated also current-voltage-luminescence characteristics for the new top-emitting organic light-emitting diodes with different thicknesses of CuPc layer.

Visualization of Neutrophil Extracellular Traps and Fibrin Meshwork in Human Fibrinopurulent Inflammatory Lesions: III. Correlative Light and Electron Microscopic Study

PubMed Central

Onouchi, Takanori; Shiogama, Kazuya; Mizutani, Yasuyoshi; Takaki, Takashi; Tsutsumi, Yutaka

2016-01-01

Neutrophil extracellular traps (NETs) released from dead neutrophils at the site of inflammation represent webs of neutrophilic DNA stretches dotted with granule-derived antimicrobial proteins, including lactoferrin, and play important roles in innate immunity against microbial infection. We have shown the coexistence of NETs and fibrin meshwork in varied fibrinopurulent inflammatory lesions at both light and electron microscopic levels. In the present study, correlative light and electron microscopy (CLEM) employing confocal laser scanning microscopy and scanning electron microscopy was performed to bridge light and electron microscopic images of NETs and fibrin fibrils in formalin-fixed, paraffin-embedded, autopsied lung sections of legionnaire’s pneumonia. Lactoferrin immunoreactivity and 4'-6-diamidino-2-phenylindole (DAPI) reactivity were used as markers of NETs, and fibrin was probed by fibrinogen gamma chain. Of note is that NETs light microscopically represented as lactoferrin and DAPI-colocalized dots, 2.5 μm in diameter. CLEM gave super-resolution images of NETs and fibrin fibrils: “Dotted” NETs were ultrastructurally composed of fine filaments and masses of 58 nm-sized globular materials. A fibrin fibril consisted of clusters of smooth-surfaced filaments. NETs filaments (26 nm in diameter) were significantly thinner than fibrin filaments (295 nm in diameter). Of note is that CLEM was applicable to formalin-fixed, paraffin-embedded sections of autopsy material. PMID:27917008

Multiscale tomographic analysis of heterogeneous cast Al-Si-X alloys.

PubMed

Asghar, Z; Requena, G; Sket, F

2015-07-01

The three-dimensional microstructure of cast AlSi12Ni and AlSi10Cu5Ni2 alloys is investigated by laboratory X-ray computed tomography, synchrotron X-ray computed microtomography, light optical tomography and synchrotron X-ray computed microtomography with submicrometre resolution. The results obtained with each technique are correlated with the size of the scanned volumes and resolved microstructural features. Laboratory X-ray computed tomography is sufficient to resolve highly absorbing aluminides but eutectic and primary Si remain unrevealed. Synchrotron X-ray computed microtomography at ID15/ESRF gives better spatial resolution and reveals primary Si in addition to aluminides. Synchrotron X-ray computed microtomography at ID19/ESRF reveals all the phases ≥ ∼1 μm in volumes about 80 times smaller than laboratory X-ray computed tomography. The volumes investigated by light optical tomography and submicrometre synchrotron X-ray computed microtomography are much smaller than laboratory X-ray computed tomography but both techniques provide local chemical information on the types of aluminides. The complementary techniques applied enable a full three-dimensional characterization of the microstructure of the alloys at length scales ranging over six orders of magnitude. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Efficacy of oral exfoliative cytology in diabetes mellitus patients: a light microscopic and confocal microscopic study.

PubMed

Gopal, Deepika; Malathi, N; Reddy, B Thirupathi

2015-03-01

Diabetes mellitus (DM) has become a global problem. By monitoring the health status of these individuals, diabetic complications can be prevented. We aimed to analyze alterations in the morphology and cytomorphometry of buccal epithelial cells of type 2 DM patients using oral exfoliative cytology technique and determine its importance in public health screening, diagnosis and monitoring of diabetes mellitus. The study was carried out in 100 type 2 DM patients and 30 healthy individuals. Smears were taken from the right buccal mucosa and stained by the Papanicolaou technique. Staining with Acridine orange was carried out to view qualitative changes with confocal laser scanning microscope (LSM-510 Meta). The cytomorphometry was evaluated using IMAGE PRO PLUS 5.5 software with Evolution LC camera. All findings were statistically analyzed. The results showed that with increase in fasting plasma glucose levels, there is significant increase in nuclear area, decrease in cytoplasmic area, and increase in nuclear cytoplasmic ratio (p < 0.05) when compared to the control group. Various qualitative changes were noted, such as cell degeneration, micronuclei, binucleation, intracytoplasmic inclusion, candida and keratinization. In the present study, we found significant alterations in the cytomorphometry and cytomorphology of buccal epithelial cells of type 2 DM patients. This study supports and extends the view that these cellular changes can alert the clinician to the possibility of diabetes and aid in monitoring of diabetes throughout the lifetime of the patient.

Time-gated luminescence microscopy allowing direct visual inspection of lanthanide-stained microorganisms in background-free condition.

PubMed

Jin, Dayong; Piper, James A

2011-03-15

Application of standard immuno-fluorescence microscopy techniques for detection of rare-event microorganisms in dirty samples is severely limited by autofluorescence of nontarget organisms or other debris. Time-gated detection using gateable array detectors in combination with microsecond-lifetime luminescent bioprobes (usually lanthanide-based) is highly effective in suppression of (nanosecond-lifetime) autofluorescence background; however, the complexity and cost of the instrumentation is a major barrier to application of these techniques to routine diagnostics. We report a practical, low-cost implementation of time-gated luminescence detection in a standard epifluorescence microscope which has been modified to include a high-power pulsed UV light-emitting diode (LED) illumination source and a standard fast chopper inserted in the focal plane behind a microscope eyepiece. Synchronization of the pulsed illumination/gated detection cycle is driven from the clock signal from the chopper. To achieve time-gated luminescence intensities sufficient for direct visual observation, we use high cycle rates, up to 2.5 kHz, taking advantage of the fast switching capabilities of the LED source. We have demonstrated real-time direct-visual inspection of europium-labeled Giardia lamblia cysts in dirty samples and Cryptosporidium parvum oocysts in fruit juice concentrate. The signal-to-background ratio has been enhanced by a factor of 18 in time-gated mode. The availability of low-cost, robust time-gated microscopes will aid development of long-lifetime luminescence bioprobes and accelerate their application in routine laboratory diagnostics.

Williams configures the LMM

NASA Image and Video Library

2016-04-18

ISS047e066551 (04/18/2016) --- NASA astronaut Jeff Williams configures the station’s Light Microscopy Module (LMM), a modified commercial, highly flexible, state-of-the-art light imaging microscope facility that provides researchers with powerful diagnostic hardware and software. The LMM enables novel research of microscopic phenomena in microgravity, with the capability of remotely acquiring and downloading digital images and videos across many levels of magnification.

A versatile new technique to clear mouse and human brain

NASA Astrophysics Data System (ADS)

Costantini, Irene; Di Giovanna, Antonino Paolo; Allegra Mascaro, Anna Letizia; Silvestri, Ludovico; Müllenbroich, Marie Caroline; Sacconi, Leonardo; Pavone, Francesco S.

2015-07-01

Large volumes imaging with microscopic resolution is limited by light scattering. In the last few years based on refractive index matching, different clearing approaches have been developed. Organic solvents and water-based optical clearing agents have been used for optical clearing of entire mouse brain. Although these methods guarantee high transparency and preservation of the fluorescence, though present other non-negligible limitations. Tissue transformation by CLARITY allows high transparency, whole brain immunolabelling and structural and molecular preservation. This method however requires a highly expensive refractive index matching solution limiting practical applicability. In this work we investigate the effectiveness of a water-soluble clearing agent, the 2,2'-thiodiethanol (TDE) to clear mouse and human brain. TDE does not quench the fluorescence signal, is compatible with immunostaining and does not introduce any deformation at sub-cellular level. The not viscous nature of the TDE make it a suitable agent to perform brain slicing during serial two-photon (STP) tomography. In fact, by improving penetration depth it reduces tissue slicing, decreasing the acquisition time and cutting artefacts. TDE can also be used as a refractive index medium for CLARITY. The potential of this method has been explored by imaging a whole transgenic mouse brain with the light sheet microscope. Moreover we apply this technique also on blocks of dysplastic human brain tissue transformed with CLARITY and labeled with different antibody. This clearing approach significantly expands the application of single and two-photon imaging, providing a new useful method for quantitative morphological analysis of structure in mouse and human brain.

Studying aerosol light scattering based on aspect ratio distribution observed by fluorescence microscope.

PubMed

Li, Li; Zheng, Xu; Li, Zhengqiang; Li, Zhanhua; Dubovik, Oleg; Chen, Xingfeng; Wendisch, Manfred

2017-08-07

Particle shape is crucial to the properties of light scattered by atmospheric aerosol particles. A method of fluorescence microscopy direct observation was introduced to determine the aspect ratio distribution of aerosol particles. The result is comparable with that of the electron microscopic analysis. The measured aspect ratio distribution has been successfully applied in modeling light scattering and further in simulation of polarization measurements of the sun/sky radiometer. These efforts are expected to improve shape retrieval from skylight polarization by using directly measured aspect ratio distribution.

Light Microscopy Microscope Experiment

NASA Image and Video Library

2016-02-04

Ground testing for the first confocal Light Microscopy Microscope (LMM) Experiment. Procter and Gamble is working with NASA Glenn scientists to prepare for a study that examines product stabilizers in a microgravity environment. The particles in the tube glow orange because they have been fluorescently tagged with a dye that reacts to green laser lights to allow construction of a 3D image point by point. The experiment, which will be sent to the ISS later this year, will help P&G develop improved product stabilizers to extend shelf life and develop more environmentally friendly packaging.

Reconstruction of explicit structural properties at the nanoscale via spectroscopic microscopy

NASA Astrophysics Data System (ADS)

Cherkezyan, Lusik; Zhang, Di; Subramanian, Hariharan; Taflove, Allen; Backman, Vadim

2016-02-01

The spectrum registered by a reflected-light bright-field spectroscopic microscope (SM) can quantify the microscopically indiscernible, deeply subdiffractional length scales within samples such as biological cells and tissues. Nevertheless, quantification of biological specimens via any optical measures most often reveals ambiguous information about the specific structural properties within the studied samples. Thus, optical quantification remains nonintuitive to users from the diverse fields of technique application. In this work, we demonstrate that the SM signal can be analyzed to reconstruct explicit physical measures of internal structure within label-free, weakly scattering samples: characteristic length scale and the amplitude of spatial refractive-index (RI) fluctuations. We present and validate the reconstruction algorithm via finite-difference time-domain solutions of Maxwell's equations on an example of exponential spatial correlation of RI. We apply the validated algorithm to experimentally measure structural properties within isolated cells from two genetic variants of HT29 colon cancer cell line as well as within a prostate tissue biopsy section. The presented methodology can lead to the development of novel biophotonics techniques that create two-dimensional maps of explicit structural properties within biomaterials: the characteristic size of macromolecular complexes and the variance of local mass density.

Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

PubMed

Vinegoni, Claudio; Fumene Feruglio, Paolo; Brand, Christian; Lee, Sungon; Nibbs, Antoinette E; Stapleton, Shawn; Shah, Sunil; Gryczynski, Ignacy; Reiner, Thomas; Mazitschek, Ralph; Weissleder, Ralph

2017-07-01

The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

Microscopic image processing systems for measuring nonuniform film thickness profiles

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

Liu, A.H.; Plawsky, J.L.; DasGupta, S.

1994-01-01

In very thin liquid films. transport processes are controlled by the temperature and the interfacial intermolecular force field which is a function of the film thickness profile and interfacial properties. The film thickness profile and interfacial properties can be measured most efficiently using a microscopic image processing system. IPS, to record the intensity pattern of the reflected light from the film. There are two types of IPS: an image analyzing interferometer (IAI) and/or an image scanning ellipsometer (ISE). The ISE is a novel technique to measure the two dimensional thickness profile of a nonuniform, thin film, from 1 nm upmore » to several {mu}m, in a steady state as well as in a transient state. It is a full field imaging technique which can study every point on the surface simultaneously with high spatial resolution and thickness sensitivity, i.e., it can measure and map the 2-D film thickness profile. Using the ISE, the transient thickness profile of a draining thin liquid film was measured and modeled. The interfacial conditions were determined in situ by measuring the Hamaker constant. The ISE and IAI systems are compared.« less

The Potential Protective Effects of 2-aminoethyl Diphenylborinate against Inner Ear Acoustic Trauma: Experimental Study Using Transmission and Scanning Electron Microscopy.

PubMed

Kaymakçı, Mustafa; Acar, Mustafa; Burukoglu, Dilek; Kutlu, Hatice Mehtap; Shojaolsadati, Paria; Cingi, Cemal; Bayar Muluk, Nuray

2015-04-01

In this prospective experimental study, we investigated the preventive effects of 2-aminoethyl diphenylborinate (2-APB) in rats exposed to acoustic trauma (AT). Light microscopic, transmission electron microscopic (TEM), and scanning electron microscopic (SEM) examinations were performed. Eighteen healthy Wistar albino rats were divided into the following three groups: groups 1 (control), 2 (AT), and 3 (AT+APB). The rats in groups 2 and 3 were exposed to AT; in group 3 rats, 2-APB at 2 mg/kg was also administered, initially transperitoneally, after 10 min. During the light microscopic, TEM, and SEM examinations, the structures of the cochlear hair cells, stereocilia, and Deiter's cells were normal in the control group. In the AT group, the organ of Corti and proximate structures were damaged according to the light microscopic examination. During the TEM examination, intense cellular damage and stereocilia loss were detected, while during the SEM examination, extensive damage and stereocilia loss were observed. Decreased damage with preserved cochlear structure was detected during the light microscopic examination in the AT+APB group than in the AT group. During the TEM and SEM examinations, although stereocilia loss occurred in the AT+APB group, near-normal cell, cilia, and tectorial membrane structures were also observed in the AT+APB group compared with the AT group. 2-APB may have protective effects against AT damage of the cochlea. The main mechanism underlying this effect is the inhibition of the vasoconstriction of the cochlear spiral modiolar artery, thereby improving cochlear blood flow. We conclude that 2-APB may also be effective if used immediately following AT.

Laser speckle contrast imaging using light field microscope approach

NASA Astrophysics Data System (ADS)

Ma, Xiaohui; Wang, Anting; Ma, Fenghua; Wang, Zi; Ming, Hai

2018-01-01

In this paper, a laser speckle contrast imaging (LSCI) system using light field (LF) microscope approach is proposed. As far as we known, it is first time to combine LSCI with LF. To verify this idea, a prototype consists of a modified LF microscope imaging system and an experimental device was built. A commercially used Lytro camera was modified for microscope imaging. Hollow glass tubes with different depth fixed in glass dish were used to simulate the vessels in brain and test the performance of the system. Compared with conventional LSCI, three new functions can be realized by using our system, which include refocusing, extending the depth of field (DOF) and gathering 3D information. Experiments show that the principle is feasible and the proposed system works well.

Lensfree microscopy on a cellphone

PubMed Central

Tseng, Derek; Mudanyali, Onur; Oztoprak, Cetin; Isikman, Serhan O.; Sencan, Ikbal; Yaglidere, Oguzhan; Ozcan, Aydogan

2010-01-01

We demonstrate lensfree digital microscopy on a cellphone. This compact and light-weight holographic microscope installed on a cellphone does not utilize any lenses, lasers or other bulky optical components and it may offer a cost-effective tool for telemedicine applications to address various global health challenges. Weighing ~38 grams (<1.4 ounces), this lensfree imaging platform can be mechanically attached to the camera unit of a cellphone where the samples are loaded from the side, and are vertically illuminated by a simple light-emitting diode (LED). This incoherent LED light is then scattered from each micro-object to coherently interfere with the background light, creating the lensfree hologram of each object on the detector array of the cellphone. These holographic signatures captured by the cellphone permit reconstruction of microscopic images of the objects through rapid digital processing. We report the performance of this lensfree cellphone microscope by imaging various sized micro-particles, as well as red blood cells, white blood cells, platelets and a waterborne parasite (Giardia lamblia). PMID:20445943

Digital photography for the light microscope: results with a gated, video-rate CCD camera and NIH-image software.

PubMed

Shaw, S L; Salmon, E D; Quatrano, R S

1995-12-01

In this report, we describe a relatively inexpensive method for acquiring, storing and processing light microscope images that combines the advantages of video technology with the powerful medium now termed digital photography. Digital photography refers to the recording of images as digital files that are stored, manipulated and displayed using a computer. This report details the use of a gated video-rate charge-coupled device (CCD) camera and a frame grabber board for capturing 256 gray-level digital images from the light microscope. This camera gives high-resolution bright-field, phase contrast and differential interference contrast (DIC) images but, also, with gated on-chip integration, has the capability to record low-light level fluorescent images. The basic components of the digital photography system are described, and examples are presented of fluorescence and bright-field micrographs. Digital processing of images to remove noise, to enhance contrast and to prepare figures for printing is discussed.

Total variation based image deconvolution for extended depth-of-field microscopy images

NASA Astrophysics Data System (ADS)

Hausser, F.; Beckers, I.; Gierlak, M.; Kahraman, O.

2015-03-01

One approach for a detailed understanding of dynamical cellular processes during drug delivery is the use of functionalized biocompatible nanoparticles and fluorescent markers. An appropriate imaging system has to detect these moving particles so as whole cell volumes in real time with high lateral resolution in a range of a few 100 nm. In a previous study Extended depth-of-field microscopy (EDF-microscopy) has been applied to fluorescent beads and tradiscantia stamen hair cells and the concept of real-time imaging has been proved in different microscopic modes. In principle a phase retardation system like a programmable space light modulator or a static waveplate is incorporated in the light path and modulates the wavefront of light. Hence the focal ellipsoid is smeared out and images seem to be blurred in a first step. An image restoration by deconvolution using the known point-spread-function (PSF) of the optical system is necessary to achieve sharp microscopic images of an extended depth-of-field. This work is focused on the investigation and optimization of deconvolution algorithms to solve this restoration problem satisfactorily. This inverse problem is challenging due to presence of Poisson distributed noise and Gaussian noise, and since the PSF used for deconvolution exactly fits in just one plane within the object. We use non-linear Total Variation based image restoration techniques, where different types of noise can be treated properly. Various algorithms are evaluated for artificially generated 3D images as well as for fluorescence measurements of BPAE cells.

Quantum coherent optical phase modulation in an ultrafast transmission electron microscope.

PubMed

Feist, Armin; Echternkamp, Katharina E; Schauss, Jakob; Yalunin, Sergey V; Schäfer, Sascha; Ropers, Claus

2015-05-14

Coherent manipulation of quantum systems with light is expected to be a cornerstone of future information and communication technology, including quantum computation and cryptography. The transfer of an optical phase onto a quantum wavefunction is a defining aspect of coherent interactions and forms the basis of quantum state preparation, synchronization and metrology. Light-phase-modulated electron states near atoms and molecules are essential for the techniques of attosecond science, including the generation of extreme-ultraviolet pulses and orbital tomography. In contrast, the quantum-coherent phase-modulation of energetic free-electron beams has not been demonstrated, although it promises direct access to ultrafast imaging and spectroscopy with tailored electron pulses on the attosecond scale. Here we demonstrate the coherent quantum state manipulation of free-electron populations in an electron microscope beam. We employ the interaction of ultrashort electron pulses with optical near-fields to induce Rabi oscillations in the populations of electron momentum states, observed as a function of the optical driving field. Excellent agreement with the scaling of an equal-Rabi multilevel quantum ladder is obtained, representing the observation of a light-driven 'quantum walk' coherently reshaping electron density in momentum space. We note that, after the interaction, the optically generated superposition of momentum states evolves into a train of attosecond electron pulses. Our results reveal the potential of quantum control for the precision structuring of electron densities, with possible applications ranging from ultrafast electron spectroscopy and microscopy to accelerator science and free-electron lasers.

Quantum coherent optical phase modulation in an ultrafast transmission electron microscope

NASA Astrophysics Data System (ADS)

Feist, Armin; Echternkamp, Katharina E.; Schauss, Jakob; Yalunin, Sergey V.; Schäfer, Sascha; Ropers, Claus

2015-05-01

Coherent manipulation of quantum systems with light is expected to be a cornerstone of future information and communication technology, including quantum computation and cryptography. The transfer of an optical phase onto a quantum wavefunction is a defining aspect of coherent interactions and forms the basis of quantum state preparation, synchronization and metrology. Light-phase-modulated electron states near atoms and molecules are essential for the techniques of attosecond science, including the generation of extreme-ultraviolet pulses and orbital tomography. In contrast, the quantum-coherent phase-modulation of energetic free-electron beams has not been demonstrated, although it promises direct access to ultrafast imaging and spectroscopy with tailored electron pulses on the attosecond scale. Here we demonstrate the coherent quantum state manipulation of free-electron populations in an electron microscope beam. We employ the interaction of ultrashort electron pulses with optical near-fields to induce Rabi oscillations in the populations of electron momentum states, observed as a function of the optical driving field. Excellent agreement with the scaling of an equal-Rabi multilevel quantum ladder is obtained, representing the observation of a light-driven `quantum walk' coherently reshaping electron density in momentum space. We note that, after the interaction, the optically generated superposition of momentum states evolves into a train of attosecond electron pulses. Our results reveal the potential of quantum control for the precision structuring of electron densities, with possible applications ranging from ultrafast electron spectroscopy and microscopy to accelerator science and free-electron lasers.

Orientation and phase mapping in the transmission electron microscope using precession-assisted diffraction spot recognition: state-of-the-art results.

PubMed

Viladot, D; Véron, M; Gemmi, M; Peiró, F; Portillo, J; Estradé, S; Mendoza, J; Llorca-Isern, N; Nicolopoulos, S

2013-10-01

A recently developed technique based on the transmission electron microscope, which makes use of electron beam precession together with spot diffraction pattern recognition now offers the possibility to acquire reliable orientation/phase maps with a spatial resolution down to 2 nm on a field emission gun transmission electron microscope. The technique may be described as precession-assisted crystal orientation mapping in the transmission electron microscope, precession-assisted crystal orientation mapping technique-transmission electron microscope, also known by its product name, ASTAR, and consists in scanning the precessed electron beam in nanoprobe mode over the specimen area, thus producing a collection of precession electron diffraction spot patterns, to be thereafter indexed automatically through template matching. We present a review on several application examples relative to the characterization of microstructure/microtexture of nanocrystalline metals, ceramics, nanoparticles, minerals and organics. The strengths and limitations of the technique are also discussed using several application examples. ©2013 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.

Fluorescence microscope (Cyscope) for malaria diagnosis in pregnant women in Medani Hospital, Sudan.

PubMed

Hassan, Saad El-Din H; Haggaz, Abd Elrahium D; Mohammed-Elhassan, Ehab B; Malik, Elfatih M; Adam, Ishag

2011-09-24

Accuracy of diagnosis is the core for malaria control. Although microscopy is the gold standard in malaria diagnosis, its reliability is largely dependent on user skill. We compared performance of Cyscope fluorescence microscope with the Giemsa stained light microscopy for the diagnosis of malaria among pregnant women at Medani Hospital in Central Sudan. The area is characterized by unstable malaria transmission. Socio-demographic characteristics and obstetrics history were gathered using pre-tested questionnaires. Blood samples were collected from febrile pregnant women who were referred as malaria case following initial diagnosis by general microscopist. During the study period 128 febrile pregnant women presented at the hospital. Among them, Plasmodium falciparum malaria was detected in 82 (64.1%) and 80 (62.5%) by the Giemsa-stained light microscopy and the Cyscope fluorescence microscope, respectively. The sensitivity of the Cyscope fluorescence microscope was 97.6% (95% CI: 92.2%-99.6%). Out of 46 which were negative by Giemsa-stained light microscopy, 5 were positive by the Cyscope fluorescence microscope. This is translated in specificity of 89.1% (95% CI: 77.5%-95.9%). The positive and negative predictive value of Cyscope fluorescence microscope was 94.1% (95% CI: 87.4% -97.8%) and 95.3% (95% CI: 85.4% - 99.2%), respectively. This study has shown that Cyscope fluorescence microscope is a reliable diagnostic, sensitive and specific in diagnosing P. falciparum malaria among pregnant women in this setting. Further studies are needed to determine effectiveness in diagnosing other Plasmodium species and to compare it with other diagnostic tools e.g. rapid diagnostic tests and PCR.

Early quantitative method for measuring germination in non-green spores of Dryopteris paleacea using an epifluorescence-microscope technique

NASA Technical Reports Server (NTRS)

Scheuerlein, R.; Wayne, R.; Roux, S. J.

1988-01-01

A method is described to determine germination by blue-light excited red fluorescence in the positively photoblastic spores of Dryopteris paleacea Sw. This fluorescence is due to chlorophyll as evidenced from 1) a fluorescence-emission spectrum in vivo, where a bright fluorescence around 675 nm is obtained only in red light (R)-irradiated spores and 2) in vitro measurements with acetone extracts prepared from homogenized spores. Significant amounts of chlorophyll can be found only in R-treated spores; this chlorophyll exhibits an emission band around 668 nm, when irradiated with 430 nm light at 21 degrees C. Compared to other criteria for germination, such as swelling of the cell, coat splitting, greening, and rhizoid formation, which require longer periods after induction for their expression, chlorophyll fluorescence can be used to quantify germination after two days. This result is confirmed by fluence-response curves for R-induced spore germination; the same relationship between applied R and germination is obtained by the evaluation with the epifluorescence method 2 days after the light treatment as compared with the evaluation with bright-field microscopy 5 days after the inducing R. Using this technique we show for the first time that Ca2+ contributes to the signal-transduction chain in phytochrome-mediated chlorophyll synthesis in spores of Dryopteris paleacea.

EDITORIAL: Nanotechnology in motion Nanotechnology in motion

NASA Astrophysics Data System (ADS)

Demming, Anna

2012-02-01

Microscopes provide tools of inimitable value for probing the building blocks of the world around us. The identity of the inventor of the first microscope remains under debate, but a name unequivocally linked with early developments in microscopy is Robert Hooke. His Micrographia published in 1665, was the first ever bestseller in science and brought topics in microscopy to the broader public eye with pages of detailed micrographs, most famously the fly's eye and plant cells. Since the first microscopes in the late 16th century, ingenious alternatives to the original optical microscopes have been developed to create images of the world at ever smaller dimensions. Innovations include scanning probe techniques such as the atomic force microscope [1]. As Toshio Ando describes in a review in this issue [2], these devices have also entered a new era in the past decade with the development of high-speed atomic force microscopy. Now, we can not only see the nanoscale components that make up the world around us, but we can watch them at work. One of the first innovations in optical microscopy was the use of dyes. This principle first came into practice with the use of ultraviolet light to reveal previously indistinguishable features. As explained by a researcher in the early 1930s, 'It is obvious that if the dyes used for selective staining in ordinary microscopical work are supplemented by substances which cause a particular detail of the structure to fluoresce with a specific colour in ultraviolet light, then many strings will be added to the bow of the practical microscopist' [3]. More recently, emphasis on the role of plasmons—collective oscillations of electrons in nanoscale metal structures—has received considerable research attention. Plasmons enhance the local electromagnetic field and can lead to increased fluorescence rates from nearby fluorophores depending on the efficiency of the counteracting process, non-radiative transfer [4]. The 1930s also saw the development of the electron microscope, which aimed to exceed the resolving power of diffraction-limited optical microscopes. Since the diffraction limit is proportional to the incident wavelength, the shorter wavelength electron beam allows smaller features to be resolved than optical light. Ernst Ruska shared the Nobel Prize for Physics in 1986 for his work in developing the transmission electron microscope [5]. The technique continues to provide an invaluable tool in nanotechnology studies, as demonstrated recently by a collaboration of researchers in the US, Singapore and Korea used electron and atomic force microscopy in their investigation of the deposition of gold nanoparticles on graphene and the enhanced conductivity of the doped film [6]. The other half of the 1986 Nobel Prize was awarded jointly to Gerd Binnig and Heinrich Rohrer 'for their design of the scanning tunnelling microscope'. The scanning tunnelling microscope offered the first glimpses of atomic scale features, galvanizing research in nanoscale science and technology into a burst of fruitful activity that persists to this day. Instead of using the diffraction and scattering of beams to 'see' nanoscale structures, the atomic force microscope developed by Binnig, Quate and Gerber in the 1980s [1] determines the surface topology 'by touch'. The device uses nanoscale changes in the forces exerted on a tip as it scans the sample surface to generate an image. As might be expected, innovations on the original atomic force microscope have now been developed achieving ever greater sensitivities for imaging soft matter without destroying it. Recent work by collaborators at the University of Bristol and the University of Glasgow used a cigar-shaped nanoparticle held in optical tweezers as the scanning tip. The technique is not diffraction limited, imparts less force on samples than contact scanning probe microscopy techniques, and allows highly curved and strongly scattering samples to be imaged [7]. In this issue, Toshio Ando from the University of Kanazawa provides an overview of developments that have allowed atomic force microscopy to move from rates of the order of one frame a minute to over a thousand frames per second in constant height mode, as reported by Mervyn Miles and colleagues at Bristol University and University College London [8]. Among the pioneers in the field, Ando's group demonstrated the ability to record the Brownian motion of myosin V molecules on mica with image capture rates of 100 x 100 pixels in 80 ms over a decade ago [9]. The developments unleash the potential of atomic force microscopy to observe the dynamics of biological and materials systems. If seeing is believing, the ability to present real motion pictures of the nanoworld cannot fail to capture the public imagination and stimulate burgeoning new avenues of scientific endeavour. Nearly 350 years on from the publication Micrographia, images in microscopy have moved from the page to the movies. References [1] Binnig G, Quate C F, and Gerber Ch 1986 Phys. Rev. Lett. 56 930-3 [2] Ando T 2012 Nanotechnology 23 062001 [3] J G 1934 Nature 134 635-6 [4] Bharadwaj P, Anger P and Novotny L 2007 Nanotechnology 18 044017 [5] The Nobel Prize in Physics 1986 Nobelprize.org [6] Kim K K, Reina A, Shi Y, Park H, Li L-J, Lee Y H and Kong J 2010 Nanotechnology 21 285205 [7] Phillips D B, Grieve J A, Olof S N, Kocher S J, Bowman R, Padgett M J, Miles M J and Carberry D M 2011 Nanotechnology 22 285503 [8] Picco L M, Bozec L, Ulcinas A, Engledew D J, Antognozzi M, Horton M A and Miles M J 2007 Nanotechnology 18 044030 [9] Ando T, Kodera N, Takai E, Maruyama D, Saito K and Toda A 2001 Proc. Natl. Acad. Sci. 98 12468

Near-field microscopy with a microfabricated solid immersion lens

NASA Astrophysics Data System (ADS)

Fletcher, Daniel Alden

2001-07-01

Diffraction of focused light prevents optical microscopes from resolving features in air smaller than half the wavelength, λ Spatial resolution can be improved by passing light through a sub-wavelength metal aperture scanned close to a sample, but aperture-based probes suffer from low optical throughput, typically below 10-4. An alternate and more efficient technique is solid immersion microscopy in which light is focused through a high refractive index Solid Immersion Lens (SIL). This work describes the fabrication, modeling, and use of a microfabricated SIL to obtain spatial resolution better than the diffraction limit in air with high optical throughput for infrared applications. SILs on the order of 10 μm in diameter are fabricated from single-crystal silicon and integrated onto silicon cantilevers with tips for scanning. We measure a focused spot size of λ/5 with optical throughput better than 10-1 at a wavelength of λ = 9.3 μm. Spatial resolution is improved to λ/10 with metal apertures fabricated directly on the tip of the silicon SIL. Microlenses have reduced spherical aberration and better transparency than large lenses but cannot be made arbitrarily small and still focus. We model the advantages and limitations of focusing in lenses close to the wavelength in diameter using an extension of Mie theory. We also investigate a new contrast mechanism unique to microlenses resulting from the decrease in field-of-view with lens diameter. This technique is shown to achieve λ/4 spatial resolution. We explore applications of the microfabricated silicon SIL for high spatial resolution thermal microscopy and biological spectroscopy. Thermal radiation is collected through the SIL from a heated surface with spatial resolution four times better than that of a diffraction- limited infrared microscope. Using a Fourier-transform infrared spectrometer, we observe absorption peaks in bacteria cells positioned at the focus of the silicon SIL.

Plasma cell quantification in bone marrow by computer-assisted image analysis.

PubMed

Went, P; Mayer, S; Oberholzer, M; Dirnhofer, S

2006-09-01

Minor and major criteria for the diagnosis of multiple meloma according to the definition of the WHO classification include different categories of the bone marrow plasma cell count: a shift from the 10-30% group to the > 30% group equals a shift from a minor to a major criterium, while the < 10% group does not contribute to the diagnosis. Plasma cell fraction in the bone marrow is therefore critical for the classification and optimal clinical management of patients with plasma cell dyscrasias. The aim of this study was (i) to establish a digital image analysis system able to quantify bone marrow plasma cells and (ii) to evaluate two quantification techniques in bone marrow trephines i.e. computer-assisted digital image analysis and conventional light-microscopic evaluation. The results were compared regarding inter-observer variation of the obtained results. Eighty-seven patients, 28 with multiple myeloma, 29 with monoclonal gammopathy of undetermined significance, and 30 with reactive plasmocytosis were included in the study. Plasma cells in H&E- and CD138-stained slides were quantified by two investigators using light-microscopic estimation and computer-assisted digital analysis. The sets of results were correlated with rank correlation coefficients. Patients were categorized according to WHO criteria addressing the plasma cell content of the bone marrow (group 1: 0-10%, group 2: 11-30%, group 3: > 30%), and the results compared by kappa statistics. The degree of agreement in CD138-stained slides was higher for results obtained using the computer-assisted image analysis system compared to light microscopic evaluation (corr.coeff. = 0.782), as was seen in the intra- (corr.coeff. = 0.960) and inter-individual results correlations (corr.coeff. = 0.899). Inter-observer agreement for categorized results (SM/PW: kappa 0.833) was in a high range. Computer-assisted image analysis demonstrated a higher reproducibility of bone marrow plasma cell quantification. This might be of critical importance for diagnosis, clinical management and prognostics when plasma cell numbers are low, which makes exact quantifications difficult.

Optimal resolution in Fresnel incoherent correlation holographic fluorescence microscopy

PubMed Central

Brooker, Gary; Siegel, Nisan; Wang, Victor; Rosen, Joseph

2011-01-01

Fresnel Incoherent Correlation Holography (FINCH) enables holograms and 3D images to be created from incoherent light with just a camera and spatial light modulator (SLM). We previously described its application to microscopic incoherent fluorescence wherein one complex hologram contains all the 3D information in the microscope field, obviating the need for scanning or serial sectioning. We now report experiments which have led to the optimal optical, electro-optic, and computational conditions necessary to produce holograms which yield high quality 3D images from fluorescent microscopic specimens. An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram. This advance dramatically improves the resolution of the FINCH system. Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging. PMID:21445140

A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability.

PubMed

Hasan, Md Mehedi; Alam, Mohammad Wajih; Wahid, Khan A; Miah, Sayem; Lukong, Kiven Erique

2016-01-01

This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size.

A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability

PubMed Central

Hasan, Md. Mehedi; Wahid, Khan A.; Miah, Sayem; Lukong, Kiven Erique

2016-01-01

This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size. PMID:27977709

The HVAC Challenges of Upgrading an Old Lab for High-end Light Microscopes

PubMed Central

Richard, R.; Martone, P.; Callahan, L.M.

2014-01-01

The University of Rochester Medical Center forms the centerpiece of the University of Rochester's health research, teaching, patient care, and community outreach missions. Within this large facility of over 5 million square feet, demolition and remodeling of existing spaces is a constant activity. With more than $145 million in federal research funding, lab space is frequently repurposed and renovated to support this work. The URMC Medical Center Facilities Organization supporting small to medium space renovations is constantly challenged and constrained by the existing mechanical infrastructure and budgets to deliver a renovated space that functions within the equipment environmental parameters. One recent project, sponsored by the URMC Shared Resources Laboratory, demonstrates these points. The URMC Light Microscopy Shared Resource Laboratory requested renovation of a 121 sq. ft. room in a 40 year old building which would enable placement of a laser capture microdissection microscope and a Pascal 5 laser scanning confocal microscope with the instruments separated by a blackout curtain. This poster discusses the engineering approach implemented to bring an older lab into the environmental specifications needed for the proper operation of the high-end light microscopes.

From Animaculum to single molecules: 300 years of the light microscope.

PubMed

Wollman, Adam J M; Nudd, Richard; Hedlund, Erik G; Leake, Mark C

2015-04-01

Although not laying claim to being the inventor of the light microscope, Antonj van Leeuwenhoek (1632-1723) was arguably the first person to bring this new technological wonder of the age properly to the attention of natural scientists interested in the study of living things (people we might now term 'biologists'). He was a Dutch draper with no formal scientific training. From using magnifying glasses to observe threads in cloth, he went on to develop over 500 simple single lens microscopes (Baker & Leeuwenhoek 1739 Phil. Trans. 41, 503-519. (doi:10.1098/rstl.1739.0085)) which he used to observe many different biological samples. He communicated his finding to the Royal Society in a series of letters (Leeuwenhoek 1800 The select works of Antony Van Leeuwenhoek, containing his microscopical discoveries in many of the works of nature, vol. 1) including the one republished in this edition of Open Biology. Our review here begins with the work of van Leeuwenhoek before summarizing the key developments over the last ca 300 years, which has seen the light microscope evolve from a simple single lens device of van Leeuwenhoek's day into an instrument capable of observing the dynamics of single biological molecules inside living cells, and to tracking every cell nucleus in the development of whole embryos and plants.

Novel Concept of Attaching Endoscope Holder to Microscope for Two Handed Endoscopic Tympanoplasty.

PubMed

Khan, Mubarak M; Parab, Sapna R

2016-06-01

The well established techniques in tympanoplasty are routinely performed with operating microscopes for many decades now. Endoscopic ear surgeries provide minimally invasive approach to the middle ear and evolving new science in the field of otology. The disadvantage of endoscopic ear surgeries is that it is one-handed surgical technique as the non-dominant left hand of the surgeon is utilized for holding and manipulating the endoscope. This necessitated the need for development of the endoscope holder which would allow both hands of surgeon to be free for surgical manipulation and also allow alternate use of microscope during tympanoplasty. To report the preliminary utility of our designed and developed endoscope holder attachment gripping to microscope for two handed technique of endoscopic tympanoplasty. Prospective Non Randomized Clinical Study. Our endoscope holder attachment for microscope was designed and developed to aid in endoscopic ear surgery and to overcome the disadvantage of single handed endoscopic surgery. It was tested for endoscopic Tympanoplasty. The design of the endoscope holder attachment is described in detail along with its manipulation and manoeuvreing. A total of 78 endoholder assisted type 1 endoscopic cartilage tympanoplasties were operated to evaluate its feasibility for the two handed technique and to evaluate the results of endoscopic type 1 cartilage tympanoplasty. In early follow up period ranging from 6 to 20 months, the graft uptake was seen in 76 ears with one residual perforation and 1 recurrent perforations giving a success rate of 97.435 %. Our endocsope holder attachment for gripping microscope is a good option for two handed technique in endoscopic type 1 cartilage tympanoplasty. The study reports the successful application and use of our endoscope holder attachment for gripping microscope in two handed technique of endoscopic type 1 cartilage tympanoplasty and comparable results with microscopic techniques. IV.

Hybrid Al/steel-joints manufactured by ultrasound enhanced friction stir welding (USE-FSW): Process comparison, nondestructive testing and microscopic analysis

NASA Astrophysics Data System (ADS)

Thomä, M.; Wagner, G.; Straß, B.; Wolter, B.; Benfer, S.; Fürbeth, W.

2017-03-01

The process of friction stir welding (FSW) is an innovative joining technique, which proved its potential in joining dissimilar metals that are poorly fusion weldable. This ability opens a wide range for applications in industrial fields, where weight reduction by partial substitution of conventional materials through lightweight materials is a current central aim. As a consequence of this, the realization of aluminum / steel-joints is of great interest. For this material compound, several friction stir welds were carried out by different researchers for varying Al/steel-joints, whereas the definition of optimal process parameters as well as the increase of mechanical properties was in the focus of the studies. To achieve further improved properties for this dissimilar joint a newly developed hybrid process named “ultrasound enhanced friction stir welding (USE-FSW)” was applied. In this paper the resulting properties of Al/steel-joints using FSW and USE-FSW will be presented and compared. Furthermore, first results by using the nondestructive testing method “computer laminography” to analyze the developed joining area will be shown supplemented by detailed light-microscopic investigations, scanning electron microscopic analysis, and EDX.

sideSPIM – selective plane illumination based on a conventional inverted microscope

PubMed Central

Hedde, Per Niklas; Malacrida, Leonel; Ahrar, Siavash; Siryaporn, Albert; Gratton, Enrico

2017-01-01

Previously described selective plane illumination microscopy techniques typically offset ease of use and sample handling for maximum imaging performance or vice versa. Also, to reduce cost and complexity while maximizing flexibility, it is highly desirable to implement light sheet microscopy such that it can be added to a standard research microscope instead of setting up a dedicated system. We devised a new approach termed sideSPIM that provides uncompromised imaging performance and easy sample handling while, at the same time, offering new applications of plane illumination towards fluidics and high throughput 3D imaging of multiple specimen. Based on an inverted epifluorescence microscope, all of the previous functionality is maintained and modifications to the existing system are kept to a minimum. At the same time, our implementation is able to take full advantage of the speed of the employed sCMOS camera and piezo stage to record data at rates of up to 5 stacks/s. Additionally, sample handling is compatible with established methods and switching magnification to change the field of view from single cells to whole organisms does not require labor intensive adjustments of the system. PMID:29026679

Bottlenecks in bog pine multiplication by somatic embryogenesis and their visualization with the environmental scanning electron microscope.

PubMed

Vlašínová, Helena; Neděla, Vilem; Đorđević, Biljana; Havel, Ladislav

2017-07-01

Somatic embryogenesis (SE) is an important biotechnological technique used for the propagation of many pine species in vitro. However, in bog pine, one of the most endangered tree species in the Czech Republic, limitations were observed, which negatively influenced the development and further germination of somatic embryos. Although initiation frequency was very low-0.95 %, all obtained cell lines were subjected to maturation. The best responding cell line (BC1) was used and subjected to six different variants of the maturation media. The media on which the highest number of early-precotyledonary/cotyledonary somatic embryos was formed was supplemented with 121 μM abscisic acid (ABA) and with 6 % maltose. In the end of maturation experiments, different abnormalities in formation of somatic embryos were observed. For visualization and identification of abnormalities in meristem development during proliferation and maturation processes, the environmental scanning electron microscope was used. In comparison to the classical light microscope, the non-commercial environmental scanning electron microscope AQUASEM II has been found as a very useful tool for the quick recognition of apical meristem disruption and abnormal development. To our knowledge, this is the first report discussing somatic embryogenesis in bog pine. Based on this observation, the cultivation procedure could be enhanced and the method for SE of bog pine optimized.

Investigation of skin structures based on infrared wave parameter indirect microscopic imaging

NASA Astrophysics Data System (ADS)

Zhao, Jun; Liu, Xuefeng; Xiong, Jichuan; Zhou, Lijuan

2017-02-01

Detailed imaging and analysis of skin structures are becoming increasingly important in modern healthcare and clinic diagnosis. Nanometer resolution imaging techniques such as SEM and AFM can cause harmful damage to the sample and cannot measure the whole skin structure from the very surface through epidermis, dermis to subcutaneous. Conventional optical microscopy has the highest imaging efficiency, flexibility in onsite applications and lowest cost in manufacturing and usage, but its image resolution is too low to be accepted for biomedical analysis. Infrared parameter indirect microscopic imaging (PIMI) uses an infrared laser as the light source due to its high transmission in skins. The polarization of optical wave through the skin sample was modulated while the variation of the optical field was observed at the imaging plane. The intensity variation curve of each pixel was fitted to extract the near field polarization parameters to form indirect images. During the through-skin light modulation and image retrieving process, the curve fitting removes the blurring scattering from neighboring pixels and keeps only the field variations related to local skin structures. By using the infrared PIMI, we can break the diffraction limit, bring the wide field optical image resolution to sub-200nm, in the meantime of taking advantage of high transmission of infrared waves in skin structures.

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

Analysis of thin-film polymers using attenuated total internal reflection-Raman microspectroscopy.

PubMed

Tran, Willie; Tisinger, Louis G; Lavalle, Luis E; Sommer, André J

2015-01-01

Two methods commonly employed for molecular surface analysis and thin-film analysis of microscopic areas are attenuated total reflection infrared (ATR-IR) microspectroscopy and confocal Raman microspectroscopy. In the former method, the depth of the evanescent probe beam can be controlled by the wavelength of light, the angle of incidence, or the refractive index of the internal reflection element. Because the penetration depth is proportional to the wavelength of light, one could interrogate a smaller film thickness by moving from the mid-infrared region to the visible region employing Raman spectroscopy. The investigation of ATR Raman microspectroscopy, a largely unexplored technique available to Raman microspectroscopy, was carried out. A Renishaw inVia Raman microscope was externally modified and used in conjunction with a solid immersion lens (SIL) to perform ATR Raman experiments. Thin-film polymer samples were analyzed to explore the theoretical sampling depth for experiments conducted without the SIL, with the SIL, and with the SIL using evanescent excitation. The feasibility of micro-ATR Raman was examined by collecting ATR spectra from films whose thickness measured from 200 to 60 nm. Films of these thicknesses were present on a much thicker substrate, and features from the underlying substrate did not become visible until the thin film reached a thickness of 68 nm.

Study of Colour Model for Segmenting Mycobacterium Tuberculosis in Sputum Images

NASA Astrophysics Data System (ADS)

Kurniawardhani, A.; Kurniawan, R.; Muhimmah, I.; Kusumadewi, S.

2018-03-01

One of method to diagnose Tuberculosis (TB) disease is sputum test. The presence and number of Mycobacterium tuberculosis (MTB) in sputum are identified. The presence of MTB can be seen under light microscope. Before investigating through stained light microscope, the sputum samples are stained using Ziehl-Neelsen (ZN) stain technique. Because there is no standard procedure in staining, the appearance of sputum samples may vary either in background colour or contrast level. It increases the difficulty in segmentation stage of automatic MTB identification. Thus, this study investigated the colour models to look for colour channels of colour model that can segment MTB well in different stained conditions. The colour models will be investigated are each channel in RGB, HSV, CIELAB, YCbCr, and C-Y colour model and the clustering algorithm used is k-Means. The sputum image dataset used in this study is obtained from community health clinic in a district in Indonesia. The size of each image was set to 1600x1200 pixels which is having variation in number of MTB, background colour, and contrast level. The experiment result indicates that in all image conditions, blue, hue, Cr, and Ry colour channel can be used to segment MTB in one cluster well.

The morphology of the pineal gland of the yellow-toothed cavy (Galea Spixii Wagler, 1831) and red-rumped agouti (Dasyprocta leporina linnaeus, 1758).

PubMed

Câmara, Felipe Venceslau; Lopes, Igor Renno Guimarães; de Oliveira, Gleidson Benevides; Bezerra, Ferdinando Vinicius Fernandes; de Oliveira, Radan Elvis Matias; Oliveira Júnior, Carlos Magno; Silva, Alexandre Rodrigues; de Oliveira, Moacir Franco

2015-08-01

The pineal gland is an endocrine gland found in all mammals. This article describes the morphology of this important gland in two species of Caviideae, namely the yellow-toothed cavy and the red-rumped agouti. Ten adult animals of the two species used in current analysis were retrieved from the Center for the Multiplication of Wild Animals (CEMAS/UFERSA) and euthanized. The glands were removed and photographed in situ and ex situ. They were fixed in a paraformaldehyde solution 4% or glutaraldehyde 2.5% solution and submitted to routine histological techniques respectively for light and scanning electron microscopy. Macroscopically, the pineal gland with its elongated structure may be found between the cerebral hemispheres facing the rostral colliculi. Microscopically, pinealocytes and some glia cells were predominant. Contrastingly, to the cavy's pineal gland, a capsule covered the organ in the agouti, with the emission of incomplete septa to the interior, which divided it into two lobules. Light and scanning electron microscopes failed to show calcareous concretions in the pineal gland. Based on the topography of the cavy's and agouti's pineal gland, it may be classified as supra-callosum and ABC type. © 2015 Wiley Periodicals, Inc.

Driving the photoluminescent and structural properties of X2-Y2SiO5 by varying the dopant Dy3+ concentration towards cool WLED applications

NASA Astrophysics Data System (ADS)

Ramakrishna, G.; Nagabhushana, H.; Hareesh, K.; Sunitha, D. V.

2017-07-01

Dy3+ doped Y2SiO5 nanophosphors were synthesized by solution combustion technique using Calotropis gigantean milk latex and NaCl as fuel and flux respectively. Powder X-ray diffraction (PXRD) confirmed the formation of monoclinic X2-phase Y2SiO5 belonging to the phase group C2/c. Fourier transform infrared spectroscopy (FTIR) shows characteristic metal-oxygen (Y-O) vibration band at 721 cm-1. Transmission electron microscopic (TEM) and Scanning electron microscopic (SEM) morphological feature exhibits non-uniform almost spherical shaped nanosized particles. The photoluminescence (PL) emission peaks, recorded at 388 nm, showed radiative emissions at 483, 575 and 636 nm respectively. Judd-Ofelt (JO) analysis was carried out to estimate the radiative (AR) properties, radiative life time (τR), branching ratio (βR) and stimulated emission crossection (σλp). The CIE and CCT was estimated using McCamy empirical formula. In the beginning, the CIE co-ordinate values were lying in the light blue region. However, with increase in Dy3+ concentration the values shifted towards white region. CCT value was found to be ∼6984 K. Therefore, Y2SiO5:Dy3+ (9 mol%) can be used for cool day light and WLED applications.

Swept Field Laser Confocal Microscopy for Enhanced Spatial and Temporal Resolution in Live-Cell Imaging

PubMed Central

Castellano-Muñoz, Manuel; Peng, Anthony Wei; Salles, Felipe T.; Ricci, Anthony J.

2013-01-01

Confocal fluorescence microscopy is a broadly used imaging technique that enhances the signal-to-noise ratio by removing out of focal plane fluorescence. Confocal microscopes come with a variety of modifications depending on the particular experimental goals. Microscopes, illumination pathways, and light collection were originally focused upon obtaining the highest resolution image possible, typically on fixed tissue. More recently, live-cell confocal imaging has gained importance. Since measured signals are often rapid or transient, thus requiring higher sampling rates, specializations are included to enhance spatial and temporal resolution while maintaining tissue viability. Thus, a balance between image quality, temporal resolution, and tissue viability is needed. A subtype of confocal imaging, termed swept field confocal (SFC) microscopy, can image live cells at high rates while maintaining confocality. SFC systems can use a pinhole array to obtain high spatial resolution, similar to spinning disc systems. In addition, SFC imaging can achieve faster rates by using a slit to sweep the light across the entire image plane, thus requiring a single scan to generate an image. Coupled to a high-speed charge-coupled device camera and a laser illumination source, images can be obtained at greater than 1,000 frames per second while maintaining confocality. PMID:22831554

A Jones matrix formalism for simulating three-dimensional polarized light imaging of brain tissue.

PubMed

Menzel, M; Michielsen, K; De Raedt, H; Reckfort, J; Amunts, K; Axer, M

2015-10-06

The neuroimaging technique three-dimensional polarized light imaging (3D-PLI) provides a high-resolution reconstruction of nerve fibres in human post-mortem brains. The orientations of the fibres are derived from birefringence measurements of histological brain sections assuming that the nerve fibres—consisting of an axon and a surrounding myelin sheath—are uniaxial birefringent and that the measured optic axis is oriented in the direction of the nerve fibres (macroscopic model). Although experimental studies support this assumption, the molecular structure of the myelin sheath suggests that the birefringence of a nerve fibre can be described more precisely by multiple optic axes oriented radially around the fibre axis (microscopic model). In this paper, we compare the use of the macroscopic and the microscopic model for simulating 3D-PLI by means of the Jones matrix formalism. The simulations show that the macroscopic model ensures a reliable estimation of the fibre orientations as long as the polarimeter does not resolve structures smaller than the diameter of single fibres. In the case of fibre bundles, polarimeters with even higher resolutions can be used without losing reliability. When taking the myelin density into account, the derived fibre orientations are considerably improved. © 2015 The Author(s).

Scanning optical microscope with long working distance objective

DOEpatents

Cloutier, Sylvain G.

2010-10-19

A scanning optical microscope, including: a light source to generate a beam of probe light; collimation optics to substantially collimate the probe beam; a probe-result beamsplitter; a long working-distance, infinity-corrected objective; scanning means to scan a beam spot of the focused probe beam on or within a sample; relay optics; and a detector. The collimation optics are disposed in the probe beam. The probe-result beamsplitter is arranged in the optical paths of the probe beam and the resultant light from the sample. The beamsplitter reflects the probe beam into the objective and transmits resultant light. The long working-distance, infinity-corrected objective is also arranged in the optical paths of the probe beam and the resultant light. It focuses the reflected probe beam onto the sample, and collects and substantially collimates the resultant light. The relay optics are arranged to relay the transmitted resultant light from the beamsplitter to the detector.

Determining oxygen relaxations at an interface: A comparative study between transmission electron microscopy techniques.

PubMed

Gauquelin, N; van den Bos, K H W; Béché, A; Krause, F F; Lobato, I; Lazar, S; Rosenauer, A; Van Aert, S; Verbeeck, J

2017-10-01

Nowadays, aberration corrected transmission electron microscopy (TEM) is a popular method to characterise nanomaterials at the atomic scale. Here, atomically resolved images of nanomaterials are acquired, where the contrast depends on the illumination, imaging and detector conditions of the microscope. Visualization of light elements is possible when using low angle annular dark field (LAADF) STEM, annular bright field (ABF) STEM, integrated differential phase contrast (iDPC) STEM, negative spherical aberration imaging (NCSI) and imaging STEM (ISTEM). In this work, images of a NdGaO 3 -La 0.67 Sr 0.33 MnO 3 (NGO-LSMO) interface are quantitatively evaluated by using statistical parameter estimation theory. For imaging light elements, all techniques are providing reliable results, while the techniques based on interference contrast, NCSI and ISTEM, are less robust in terms of accuracy for extracting heavy column locations. In term of precision, sample drift and scan distortions mainly limits the STEM based techniques as compared to NCSI. Post processing techniques can, however, partially compensate for this. In order to provide an outlook to the future, simulated images of NGO, in which the unavoidable presence of Poisson noise is taken into account, are used to determine the ultimate precision. In this future counting noise limited scenario, NCSI and ISTEM imaging will provide more precise values as compared to the other techniques, which can be related to the mechanisms behind the image recording. Copyright © 2017 Elsevier B.V. All rights reserved.

Microscopic Procedures for Plant Meiosis.

ERIC Educational Resources Information Center

Braselton, James P.

1997-01-01

Describes laboratory techniques designed to familiarize students with meiosis and how microscopic preparations of meiosis are made. These techniques require the use of fresh or fixed flowers. Contains 18 references. (DDR)

Transmission electron microscope sample holder with optical features

DOEpatents

Milas, Mirko [Port Jefferson, NY; Zhu, Yimei [Stony Brook, NY; Rameau, Jonathan David [Coram, NY

2012-03-27

A sample holder for holding a sample to be observed for research purposes, particularly in a transmission electron microscope (TEM), generally includes an external alignment part for directing a light beam in a predetermined beam direction, a sample holder body in optical communication with the external alignment part and a sample support member disposed at a distal end of the sample holder body opposite the external alignment part for holding a sample to be analyzed. The sample holder body defines an internal conduit for the light beam and the sample support member includes a light beam positioner for directing the light beam between the sample holder body and the sample held by the sample support member.

Freezing cytorrhysis and critical temperature thresholds for photosystem II in the peat moss Sphagnum capillifolium.

PubMed

Buchner, Othmar; Neuner, Gilbert

2010-07-01

Leaflets of Sphagnum capillifolium were exposed to temperatures from -5 degrees C to +60 degrees C under controlled conditions while mounted on a microscope stage. The resultant cytological response to these temperature treatments was successfully monitored using a light and fluorescence microscope. In addition to the observable cytological changes during freezing cytorrhysis and heat exposure on the leaflets, the concomitant critical temperature thresholds for inactivation of photosystem II (PS II) were studied using a micro fibre optic and a chlorophyll fluorometer mounted to the microscope stage. Chlorophyllous cells of S. capillifolium showed extended freezing cytorrhysis immediately after ice nucleation at -1.1 degrees C in the water in which the leaflets were submersed during the measurement. The occurrence of freezing cytorrhysis, which was visually manifested by cell shrinkage, was highly dynamic and was completed within 2 s. A total reduction of the mean projected diameter of the chloroplast containing area during freezing cytorrhysis from 8.9 to 3.8 microm indicates a cell volume reduction of approximately -82%. Simultaneous measurement of chlorophyll fluorescence of PS II was possible even through the frozen water in which the leaf samples were submersed. Freezing cytorrhysis was accompanied by a sudden rise of basic chlorophyll fluorescence. The critical freezing temperature threshold of PS II was identical to the ice nucleation temperature (-1.1 degrees C). This is significantly above the temperature threshold at which frost damage to S. capillifolium leaflets occurs (-16.1 degrees C; LT(50)) which is higher than observed in most higher plants from the European Alps during summer. High temperature thresholds of PS II were 44.5 degrees C which is significantly below the heat tolerance of chlorophyllous cells (49.9 degrees C; LT(50)). It is demonstrated that light and fluorescence microscopic techniques combined with simultaneous chlorophyll fluorescence measurements may act as a useful tool to study heat, low temperature, and ice-encasement effects on the cellular structure and primary photosynthetic processes of intact leaf tissues.

Probing the coordination environment of Ti(3+) ions coordinated to nitrogen-containing Lewis bases.

PubMed

Morra, E; Maurelli, S; Chiesa, M; Van Doorslaer, S

2015-08-28

Multi-frequency continuous-wave and pulsed EPR techniques are employed to investigate the coordination of nitrogen-containing ligands to Ti(3+)-chloro complexes. Frozen solutions of TiCl3 and TiCl3(Py)3 dissolved in nitrogen-containing solvents have been investigated together with the TiCl3(Py)3 solid-state complex. For these different systems, the hyperfine and nuclear quadrupole data of Ti(3+)-bound (14)N nuclei are reported and discussed in the light of DFT computations, allowing for a detailed description of the microscopic structure of these systems.

Integrated circuit layer image segmentation

NASA Astrophysics Data System (ADS)

Masalskis, Giedrius; Petrauskas, Romas

2010-09-01

In this paper we present IC layer image segmentation techniques which are specifically created for precise metal layer feature extraction. During our research we used many samples of real-life de-processed IC metal layer images which were obtained using optical light microscope. We have created sequence of various image processing filters which provides segmentation results of good enough precision for our application. Filter sequences were fine tuned to provide best possible results depending on properties of IC manufacturing process and imaging technology. Proposed IC image segmentation filter sequences were experimentally tested and compared with conventional direct segmentation algorithms.

Endoscopic Optical Coherence Tomography for Clinical Gastroenterology

PubMed Central

Tsai, Tsung-Han; Fujimoto, James G.; Mashimo, Hiroshi

2014-01-01

Optical coherence tomography (OCT) is a real-time optical imaging technique that is similar in principle to ultrasonography, but employs light instead of sound waves and allows depth-resolved images with near-microscopic resolution. Endoscopic OCT allows the evaluation of broad-field and subsurface areas and can be used ancillary to standard endoscopy, narrow band imaging, chromoendoscopy, magnification endoscopy, and confocal endomicroscopy. This review article will provide an overview of the clinical utility of endoscopic OCT in the gastrointestinal tract and of recent achievements using state-of-the-art endoscopic 3D-OCT imaging systems. PMID:26852678

Coccolith and silicoflagellate stratigraphy, northern mid-Atlantic Ridge and Reykjanes Ridge, Deep Sea Drilling Project Leg 49

USGS Publications Warehouse

Bukry, David

1979-01-01

Leg 49 of the Deep Sea Drilling Project recovered 192 cores at eight drilling sites, 407 through 414 (Figure 1). Light-microscope techniques were used to study the cocoliths, silicoflagellates, and sponge spicules of 120 samples from these cores. The cocolith zonation of the samples follows Bukry (1975a), and is summarized in Figure 2. Silicoflagellate zonation, summarized in Figure 3, is explained in the text. Siliceous sponge spicules are common in many samples and are briefly discussed and illustrated. One new silicoflagellate, Distephanus sulcatus, from the Plicene of Site 407, is described.

A solvent-free coating-procedure for the improved preparation of cryostat sections in light microscope histochemistry.

PubMed

Fink, S

1992-01-01

A new technique is presented for the external stabilization of cryostat sections by spraying the specimen surfaces with an aqueous solution of poly(vinyl alcohol) before each sectioning stroke. The spray freezes upon the surface and forms a tough coating which facilitates subsequent sectioning and handling especially of difficult material. The sections are affixed upon cold glass slides covered with an improved formulation of pressure-sensitive adhesive. During further processing of the affixed sections, the PVA-coating and any surrounding supporting medium dissolve without traces in the first aqueous incubation or staining solution.

[Histochemical stains for minerals by hematoxylin-lake method].

PubMed

Miyagawa, Makoto

2013-04-01

The present study was undertaken to establish the experimental animal model by histological staining methods for minerals. After intraperitoneal injections of minerals, precipitates deposited on the surface of the liver. Liver tissues were fixed in paraformaldehyde, embedded in paraffin and cut into thin sections which were used as minerals containing standard section. Several reagents for histological stains and spectrophotometry for minerals were applied in both test-tube experiments and stainings of tissue sections to test for minerals. Hematoxylin-lake was found of capable of staining minerals in tissue. A simple technique used was described for light microscopic detection of minerals.

Microcircuit testing and fabrication, using scanning electron microscopes

NASA Technical Reports Server (NTRS)

Nicolas, D. P.

1975-01-01

Scanning electron microscopes are used to determine both user-induced damages and manufacturing defects subtle enough to be missed by conventional light microscopy. Method offers greater depth of field and increased working distances.

ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy.

PubMed

Brama, Elisabeth; Peddie, Christopher J; Wilkes, Gary; Gu, Yan; Collinson, Lucy M; Jones, Martin L

2016-12-13

In-resin fluorescence (IRF) protocols preserve fluorescent proteins in resin-embedded cells and tissues for correlative light and electron microscopy, aiding interpretation of macromolecular function within the complex cellular landscape. Dual-contrast IRF samples can be imaged in separate fluorescence and electron microscopes, or in dual-modality integrated microscopes for high resolution correlation of fluorophore to organelle. IRF samples also offer a unique opportunity to automate correlative imaging workflows. Here we present two new locator tools for finding and following fluorescent cells in IRF blocks, enabling future automation of correlative imaging. The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables 'smart collection' of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables 'smart tracking' of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes.

Atomic force microscopic imaging of Acanthamoeba castellanii and Balamuthia mandrillaris trophozoites and cysts.

PubMed

Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Ateeq, Muhammad; Raza Shah, Muhammad; Kulsoom, Huma; Khan, Naveed Ahmed

2015-01-01

Light microscopy and electron microscopy have been successfully used in the study of microbes, as well as free-living protists. Unlike light microscopy, which enables us to observe living organisms or the electron microscope which provides a two-dimensional image, atomic force microscopy provides a three-dimensional surface profile. Here, we observed two free-living amoebae, Acanthamoeba castellanii and Balamuthia mandrillaris under the phase contrast inverted microscope, transmission electron microscope and atomic force microscope. Although light microscopy was of lower magnification, it revealed functional biology of live amoebae such as motility and osmoregulation using contractile vacuoles of the trophozoite stage, but it is of limited value in defining the cyst stage. In contrast, transmission electron microscopy showed significantly greater magnification and resolution to reveal the ultra-structural features of trophozoites and cysts including intracellular organelles and cyst wall characteristics but it only produced a snapshot in time of a dead amoeba cell. Atomic force microscopy produced three-dimensional images providing detailed topographic description of shape and surface, phase imaging measuring boundary stiffness, and amplitude measurements including width, height and length of A. castellanii and B. mandrillaris trophozoites and cysts. These results demonstrate the importance of the application of various microscopic methods in the biological and structural characterization of the whole cell, ultra-structural features, as well as surface components and cytoskeleton of protist pathogens. © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.

The light environment and cellular optics of the snow alga Chlamydomonas nivalis (Bauer) Wille.

PubMed

Gorton, H L; Williams, W E; Vogelmann, T C

2001-06-01

The alga Chlamydomonas nivalis lives in a high-light, cold environment: persistent alpine snowfields. Since the algae in snow receive light from all angles, the photon fluence rate is the critical parameter for photosynthesis, but it is rarely measured. We measured photon irradiance and photon fluence rate in the snow that contained blooms of C. nivalis. On a cloudless day the photon fluence rate at the snow surface was nearly twice the photon irradiance, and it can be many times greater than the photon irradiance when the solar angle is low or the light is diffuse. Beneath the surface the photon fluence rate can be five times the photon irradiance. Photon irradiance and photon fluence rate declined exponentially with depth, approximating the Bouguer-Lambert relationship. We used an integrating sphere to measure the spectral characteristics of a monolayer of cells and microscopic techniques to examine the spectral characteristics of individual cells. Astaxanthin blocked blue light and unknown absorbers blocked UV radiation; the penetration of these wavelengths through whole cells was negligible. We extracted astaxanthin, measured absorbance on a per-cell basis and estimated that the layer of astaxanthin within cells would allow only a small percentage of the blue light to reach the chloroplast, potentially protecting the chloroplast from excessive light.

A Fast Responsive Ultraviolet Sensor from mSILAR-Processed Sn-ZnO

NASA Astrophysics Data System (ADS)

Thomas, Deepu; Vijayalakshmi, K. A.; Sadasivuni, Kishor Kumar; Thomas, Ajith; Ponnamma, Deepalekshmi; Cabibihan, John-John

2017-11-01

Microwave-assisted successive ionic layer adsorption and reaction was employed to synthesize Sn-ZnO (tin-doped zinc oxide), and its sensitivity to ultraviolet radiation is compared with zinc oxide (ZnO). The sensing films were made by the dip-coated method on an indium titanium oxide glass substrate, and the sensing performance was monitored using the 300-700 nm wavelength of UV-Vis light. Excellent sensitivity and recovery were observed for the Sn-doped ZnO sensor device, especially at 380 nm wavelength of ultraviolet (UV) light (response and recovery time 2.26 s and 8.63 s, respectively, at 5 V bias voltage). The variation in photocurrent with respect to dark and light illumination atmosphere was well illustrated based on the Schottky and inter-particle network effects. Doping of Sn on ZnO nanoparticles varied the surface roughness and crystallite size as observed from scanning electron microscopic and x-ray diffraction studies. Here, we demonstrate a simple and economical fabrication technique for designing a high-performance UV light sensor. The developed device works at room temperature with high durability and stability.

Design and demonstration of multimodal optical scanning microscopy for confocal and two-photon imaging

NASA Astrophysics Data System (ADS)

Chun, Wanhee; Do, Dukho; Gweon, Dae-Gab

2013-01-01

We developed a multimodal microscopy based on an optical scanning system in order to obtain diverse optical information of the same area of a sample. Multimodal imaging researches have mostly depended on a commercial microscope platform, easy to use but restrictive to extend imaging modalities. In this work, the beam scanning optics, especially including a relay lens, was customized to transfer broadband (400-1000 nm) lights to a sample without any optical error or loss. The customized scanning optics guarantees the best performances of imaging techniques utilizing the lights within the design wavelength. Confocal reflection, confocal fluorescence, and two-photon excitation fluorescence images were obtained, through respective implemented imaging channels, to demonstrate imaging feasibility for near-UV, visible, near-IR continuous light, and pulsed light in the scanning optics. The imaging performances for spatial resolution and image contrast were verified experimentally; the results were satisfactory in comparison with theoretical results. The advantages of customization, containing low cost, outstanding combining ability and diverse applications, will contribute to vitalize multimodal imaging researches.

Fluorescence ratiometric classifier for the detection of skin pathologies

NASA Astrophysics Data System (ADS)

Anand, Suresh; Cicchi, Riccardo; Cosci, Alessandro; Rossari, Susanna; Kapsokalyvas, Dimitrios; Baria, Enrico; Maio, Vincenza; Massi, Daniela; De Giorgi, Vincenzo; Pimpinelli, Nicola; Pavone, Francesco S.

2015-07-01

Detection of pre-malignant lesions in skin could help in reducing the 5 year patient mortality rates and greatly advancing the quality of life. Current gold standard for the detection of skin pathologies is a tissue biopsy and followed by a series of steps before it is examined under a light microscope by a pathologist. The disadvantage with this method is its invasiveness. Light based biomedical point spectroscopic techniques offers an adjunct technique to invasive tissue pathology. In this context, we have implemented a simple multiplexed ratiometric approach (F470/F560 and F510/F580) based on fluorescence at two excitation wavelengths 378 nm and 445 nm respectively. The emission profile at these excitation wavelengths showed a shift towards the longer wavelengths for melanoma when compared with normal and nevus. At both excitation wavelengths, we observed an increased intensity ratios for normal, followed by nevus and melanoma. This intensity ratios provide a good diagnostic capability in differentiating normal, nevus and melanocytic skin lesions. This method could be applied in vivo because of the simplicity involved in discriminating normal and pathological skin tissues.

Demonstration of lipofuscin and Nissl bodies in crystal violet stained sections using a fluorescence technique or pyronin Y stain.

PubMed

Terr, L I

1986-09-01

This paper presents two simple, reliable methods for identification of lipofuscin and Nissl bodies in the same section. One method shows that lipofuscin stained with crystal violet retains its ability to fluoresce and can be observed under the fluorescence microscope after the stain has faded. Fading is accompanied by a gradual increase in the intensity of the fluorescence and is complete in about 5 min. Exciting illumination from this part of the spectrum also substantially fades staining of other autofluorescing tissue elements, such as lipids. Nonfluorescing structures, such as Nissl bodies, remain stained. By changing from transillumination with tungsten light to epifluorescent illumination and vice versa, both types of structures--Nissl bodies and lipofuscin--can be identified in the same section. The second technique uses pyronin Y for staining Nissl bodies in preparations previously stained with crystal violet. Nissl bodies are stained pink but lipofuscin remains violet. Lipofuscin in these sections also remains autofluorescent after the crystal violet stain has faded under violet or near-UV light.

Effect of etching time on morphological, optical, and electronic properties of silicon nanowires.

PubMed

Nafie, Nesma; Lachiheb, Manel Abouda; Bouaicha, Mongi

2012-07-16

Owing to their interesting electronic, mechanical, optical, and transport properties, silicon nanowires (SiNWs) have attracted much attention, giving opportunities to several potential applications in nanoscale electronic, optoelectronic devices, and silicon solar cells. For photovoltaic application, a superficial film of SiNWs could be used as an efficient antireflection coating. In this work we investigate the morphological, optical, and electronic properties of SiNWs fabricated at different etching times. Characterizations of the formed SiNWs films were performed using a scanning electron microscope, ultraviolet-visible-near-infrared spectroscopy, and light-beam-induced-current technique. The latter technique was used to determine the effective diffusion length in SiNWs films. From these investigations, we deduce that the homogeneity of the SiNWs film plays a key role on the electronic properties.

Inverted light-sheet microscope for imaging mouse pre-implantation development.

PubMed

Strnad, Petr; Gunther, Stefan; Reichmann, Judith; Krzic, Uros; Balazs, Balint; de Medeiros, Gustavo; Norlin, Nils; Hiiragi, Takashi; Hufnagel, Lars; Ellenberg, Jan

2016-02-01

Despite its importance for understanding human infertility and congenital diseases, early mammalian development has remained inaccessible to in toto imaging. We developed an inverted light-sheet microscope that enabled us to image mouse embryos from zygote to blastocyst, computationally track all cells and reconstruct a complete lineage tree of mouse pre-implantation development. We used this unique data set to show that the first cell fate specification occurs at the 16-cell stage.

Neuroanatomy from Mesoscopic to Nanoscopic Scales: An Improved Method for the Observation of Semithin Sections by High-Resolution Scanning Electron Microscopy

PubMed Central

Rodríguez, José-Rodrigo; Turégano-López, Marta; DeFelipe, Javier; Merchán-Pérez, Angel

2018-01-01

Semithin sections are commonly used to examine large areas of tissue with an optical microscope, in order to locate and trim the regions that will later be studied with the electron microscope. Ideally, the observation of semithin sections would be from mesoscopic to nanoscopic scales directly, instead of using light microscopy and then electron microscopy (EM). Here we propose a method that makes it possible to obtain high-resolution scanning EM images of large areas of the brain in the millimeter to nanometer range. Since our method is compatible with light microscopy, it is also feasible to generate hybrid light and electron microscopic maps. Additionally, the same tissue blocks that have been used to obtain semithin sections can later be used, if necessary, for transmission EM, or for focused ion beam milling and scanning electron microscopy (FIB-SEM). PMID:29568263

Neuroanatomy from Mesoscopic to Nanoscopic Scales: An Improved Method for the Observation of Semithin Sections by High-Resolution Scanning Electron Microscopy.

PubMed

Rodríguez, José-Rodrigo; Turégano-López, Marta; DeFelipe, Javier; Merchán-Pérez, Angel

2018-01-01

Semithin sections are commonly used to examine large areas of tissue with an optical microscope, in order to locate and trim the regions that will later be studied with the electron microscope. Ideally, the observation of semithin sections would be from mesoscopic to nanoscopic scales directly, instead of using light microscopy and then electron microscopy (EM). Here we propose a method that makes it possible to obtain high-resolution scanning EM images of large areas of the brain in the millimeter to nanometer range. Since our method is compatible with light microscopy, it is also feasible to generate hybrid light and electron microscopic maps. Additionally, the same tissue blocks that have been used to obtain semithin sections can later be used, if necessary, for transmission EM, or for focused ion beam milling and scanning electron microscopy (FIB-SEM).

Multispectral digital lensless holographic microscopy: from femtosecond laser to white light LED

NASA Astrophysics Data System (ADS)

Garcia-Sucerquia, J.

2015-04-01

The use of femtosecond laser radiation and super bright white LED in digital lensless holographic microscopy is presented. For the ultrafast laser radiation two different configurations of operation of the microscope are presented and the dissimilar performance of each one analyzed. The microscope operating with a super bright white light LED in combination with optical filters shows very competitive performance as it is compared with more expensive optical sources. The broadband emission of both radiation sources allows the multispectral imaging of biological samples to obtain spectral responses and/or full color images of the microscopic specimens; sections of the head of a Drosophila melanogaster fly are imaged in this contribution. The simple, solid, compact, lightweight, and reliable architecture of digital lensless holographic microscopy operating with broadband light sources to image biological specimens exhibiting micrometer-sized details is evaluated in the present contribution.

Light-sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development.

PubMed

Girstmair, Johannes; Zakrzewski, Anne; Lapraz, François; Handberg-Thorsager, Mette; Tomancak, Pavel; Pitrone, Peter Gabriel; Simpson, Fraser; Telford, Maximilian J

2016-06-30

Selective plane illumination microscopy (SPIM a type of light-sheet microscopy) involves focusing a thin sheet of laser light through a specimen at right angles to the objective lens. As only the thin section of the specimen at the focal plane of the lens is illuminated, out of focus light is naturally absent and toxicity due to light (phototoxicity) is greatly reduced enabling longer term live imaging. OpenSPIM is an open access platform (Pitrone et al. 2013 and OpenSPIM.org) created to give new users step-by-step instructions on building a basic configuration of a SPIM microscope, which can in principle be adapted and upgraded to each laboratory's own requirements and budget. Here we describe our own experience with the process of designing, building, configuring and using an OpenSPIM for our research into the early development of the polyclad flatworm Maritigrella crozieri - a non-model animal. Our OpenSPIM builds on the standard design with the addition of two colour laser illumination for simultaneous detection of two probes/molecules and dual sided illumination, which provides more even signal intensity across a specimen. Our OpenSPIM provides high resolution 3d images and time lapse recordings, and we demonstrate the use of two colour lasers and the benefits of two color dual-sided imaging. We used our microscope to study the development of the embryo of the polyclad flatworm M. crozieri. The capabilities of our microscope are demonstrated by our ability to record the stereotypical spiral cleavage pattern of M. crozieri with high-speed multi-view time lapse imaging. 3D and 4D (3D + time) reconstruction of early development from these data is possible using image registration and deconvolution tools provided as part of the open source Fiji platform. We discuss our findings on the pros and cons of a self built microscope. We conclude that home-built microscopes, such as an OpenSPIM, together with the available open source software, such as MicroManager and Fiji, make SPIM accessible to anyone interested in having continuous access to their own light-sheet microscope. However, building an OpenSPIM is not without challenges and an open access microscope is a worthwhile, if significant, investment of time and money. Multi-view 4D microscopy is more challenging than we had expected. We hope that our experience gained during this project will help future OpenSPIM users with similar ambitions.

Laser surgery: using the carbon dioxide laser.

PubMed Central

Wright, V. C.

1982-01-01

In 1917 Einstein theorized tha through an atomic process a unique kind of electromagnetic radiation could be produced by stimulated emission. When such radiation is in the optical or infrared spectrum it is termed laser (light amplification by stimulated emission of radiation) light. A laser, a high-intensity light source, emits a nearly parallel electromagnetic beam of energy at a given wavelength that can be captured by a lens and concentrated in the focal spot. The wavelength determines how the laser will be used. The carbon dioxide laser is now successfully employed for some surgical procedures in gynecology, otorhinolaryngology, neurosurgery, and plastic and general surgery. The CO2 laser beam is directed through the viewing system of an operating microscope or through a hand-held laser component. Its basic action in tissue is thermal vaporization; it causes minimal damage to adjacent tissues. Surgeons require special training in the basic methods and techniques of laser surgery, as well as in the safety standards that must be observed. Images FIG. 5 PMID:7074503

Investigation of light scattering characteristics of individual leukocytes using three-dimensional refractive index maps

NASA Astrophysics Data System (ADS)

Sung, Kung-Bin; Lin, Yang-Hsien; Lin, Fong-jheng; Hsieh, Chao-Mao; Wu, Shang-Ju

2017-04-01

Three-dimensional (3D) refractive-index (RI) microscopy is an emerging technique suitable for live-cell imaging due to its label-free and fast 3D imaging capabilities. We have developed a common-path system to acquire 3D RI microscopic images of cells with excellent speed and stability. After obtaining 3D RI distributions of individual leukocytes, we used a 3D finite-difference time-domain tool to study light scattering properties. Backscattering spectra of lymphocytes, monocytes and neutrophils are different from each other. Backscattering spectra of lymphocytes matched well with those of homogeneous spheres as predicted by Mie theory while backscattering spectra of neutrophils are significantly more intense than those of the other two types. This suggests the possibility of classifying the three types of leukocytes based on backscattering.

Charge transport mechanism analysis of Al/CdS:Sr{sup 2+}/ITO device under dark and light

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

Datta, Joydeep; Das, Mrinmay; Dey, Arka

2016-05-06

In this study, we have synthesized CdS:Sr{sup 2+} by hydrothermal technique. Material property has been studied by X-ray diffraction (XRD), Scanning electron microscope (SEM) and UV-vis absorption spectroscopy. XRD data revealed that there are mixed phases of CdS and SrS in the synthesized sample. The optical band gap of the material was estimated as 3.15 eV from UV-vis data. The synthesized material has been applied in metal-semiconductor device and transport properties have been analyzed by measuring current–voltage characteristics under dark and light conditions at room temperature. Variation in different device parameters like ideality factor, barrier height and series resistance ofmore » Al/CdS:Sr{sup 2+}/ITO device were analyzed by using Cheung’s function.« less

Quantification of Nanoscale Density Fluctuations in Biological Cells/Tissues: Inverse Participation Ratio (IPR) Analysis of Transmission Electron Microscopy Images and Implications for Early-Stage Cancer Detection

NASA Astrophysics Data System (ADS)

Pradhan, Prabhakar; Damania, Dhwanil; Joshi, Hrushikesh; Taflove, Allen; Roy, Hemant; Dravid, Vinayak; Backman, Vadim

2010-03-01

We report a study of the nanoscale mass density fluctuations of biological cells and tissues by quantifying their nanoscale light-localization properties. Transmission electron microscope (TEM) images of human cells and tissues are used to construct corresponding effective disordered optical lattices. Light-localization properties are studied by statistical analysis of the inverse participation ratio (IPR) of the eigenfunctions of these optical lattices at the nanoscales. Our results indicate elevation of the nanoscale disorder strength (e.g., refractive index fluctuations) in early carcinogenesis. Importantly, our results demonstrate that the increase in the nanoscale disorder represents the earliest structural alteration in cells undergoing carcinogenesis known to-date. Potential applications of the technique for early stage cancer detection will be discussed.

In vivo bioluminescence and reflectance imaging of multiple organs in bioluminescence reporter mice by bundled-fiber-coupled microscopy

PubMed Central

Ando, Yoriko; Sakurai, Takashi; Koida, Kowa; Tei, Hajime; Hida, Akiko; Nakao, Kazuki; Natsume, Mistuo; Numano, Rika

2016-01-01

Bioluminescence imaging (BLI) is used in biomedical research to monitor biological processes within living organisms. Recently, fiber bundles with high transmittance and density have been developed to detect low light with high resolution. Therefore, we have developed a bundled-fiber-coupled microscope with a highly sensitive cooled-CCD camera that enables the BLI of organs within the mouse body. This is the first report of in vivo BLI of the brain and multiple organs in luciferase-reporter mice using bundled-fiber optics. With reflectance imaging, the structures of blood vessels and organs can be seen clearly with light illumination, and it allowed identification of the structural details of bioluminescence images. This technique can also be applied to clinical diagnostics in a low invasive manner. PMID:27231601

A Fluorescence Recovery After Photobleaching (FRAP) Technique for the Measurement of Solute Transport Across Surfactant-Laden Interfaces

NASA Technical Reports Server (NTRS)

Browne, Edward P.; Hatton, T. Alan

1996-01-01

The technique of Fluorescence Recovery After Photobleaching (FRAP) has been applied to the measurement of interfacial transport in two-phase systems. FRAP exploits the loss of fluorescence exhibited by certain fluorophores when over-stimulated (photobleached), so that a two-phase system, originally at equilibrium, can be perturbed without disturbing the interface by strong light from an argon-ion laser and its recovery monitored by a microscope-mounted CCD camera as it relaxes to a new equilibrium. During this relaxation, the concentration profiles of the probe solute are measured on both sides of the interface as a function of time, yielding information about the transport characteristics of the system. To minimize the size of the meniscus between the two phases, a photolithography technique is used to selectively treat the glass walls of the cell in which the phases are contained. This allows concentration measurements to be made very close to the interface and increases the sensitivity of the FRAP technique.

The interfacial strength of carbon nanofiber epoxy composite using single fiber pullout experiments.

PubMed

Manoharan, M P; Sharma, A; Desai, A V; Haque, M A; Bakis, C E; Wang, K W

2009-07-22

Carbon nanotubes and nanofibers are extensively researched as reinforcing agents in nanocomposites for their multifunctionality, light weight and high strength. However, it is the interface between the nanofiber and the matrix that dictates the overall properties of the nanocomposite. The current trend is to measure elastic properties of the bulk nanocomposite and then compare them with theoretical models to extract the information on the interfacial strength. The ideal experiment is single fiber pullout from the matrix because it directly measures the interfacial strength. However, the technique is difficult to apply to nanocomposites because of the small size of the fibers and the requirement for high resolution force and displacement sensing. We present an experimental technique for measuring the interfacial strength of nanofiber-reinforced composites using the single fiber pullout technique and demonstrate the technique for a carbon nanofiber-reinforced epoxy composite. The experiment is performed in situ in a scanning electron microscope and the interfacial strength for the epoxy composite was measured to be 170 MPa.

Quantitative phase imaging of biological cells and tissues using singleshot white light interference microscopy and phase subtraction method for extended range of measurement

NASA Astrophysics Data System (ADS)

Mehta, Dalip Singh; Sharma, Anuradha; Dubey, Vishesh; Singh, Veena; Ahmad, Azeem

2016-03-01

We present a single-shot white light interference microscopy for the quantitative phase imaging (QPI) of biological cells and tissues. A common path white light interference microscope is developed and colorful white light interferogram is recorded by three-chip color CCD camera. The recorded white light interferogram is decomposed into the red, green and blue color wavelength component interferograms and processed it to find out the RI for different color wavelengths. The decomposed interferograms are analyzed using local model fitting (LMF)" algorithm developed for reconstructing the phase map from single interferogram. LMF is slightly off-axis interferometric QPI method which is a single-shot method that employs only a single image, so it is fast and accurate. The present method is very useful for dynamic process where path-length changes at millisecond level. From the single interferogram a wavelength-dependent quantitative phase imaging of human red blood cells (RBCs) are reconstructed and refractive index is determined. The LMF algorithm is simple to implement and is efficient in computation. The results are compared with the conventional phase shifting interferometry and Hilbert transform techniques.

Omnidirectional anti-reflection properties of vertically align SiO2 nanorod films prepared by electron beam evaporation with glancing angle deposition

NASA Astrophysics Data System (ADS)

Prachachet, R.; Samransuksamer, B.; Horprathum, M.; Eiamchai, P.; Limwichean, S.; Chananonnawathorn, C.; Lertvanithphol, T.; Muthitamongkol, P.; Boonruang, S.; Buranasiri, P.

2018-03-01

Omnidirectional anti-reflection coating nanostructure film have attracted enormous attention for the developments of the optical coating, lenses, light emitting diode, display and photovoltaic. However, fabricated of the omnidirectional antireflection nanostructure film on glass substrate in large area was a challenge topic. In the past two decades, the invention of glancing angle deposition technique as a growth of well-controlled two and three-dimensional morphologies has gained significant attention because of it is simple, fast, cost-effective and high mass production capability. In this present work, the omnidirectional anti-reflection nanostructure coating namely silicon dioxide (SiO2) nanorods has been investigated for optimized high transparent layer at all light incident angle. The SiO2 nanorod films of an optimally low refractive index have been fabricated by electron beam evaporation with the glancing angle deposition technique. The morphological of the prepared sampled were characterized by field-emission scanning electron microscope (FE-SEM) and high-resolution transmission electron microscope (HRTEM). The optical transmission and omnidirectional property of the SiO2 nanorod films were investigated by UV-Vis-NIR spectrophotometer. The measurement were performed at normal incident angle and a full spectral range of 200 - 2000 nm. The angle dependent transmission measure were investigated by rotating the specimen, with incidence angle defined relative to the surface normal of the prepared samples. The morphological characterization results showed that when the glancing angle deposition technique was applied, the vertically align SiO2 nanorods with partially isolated columnar structure can be constructed due to the enhanced shadowing and limited addtom diffusion effect. The average transmission of the vertically align SiO2 nanorods were higher than the glass substrate reference sample over the visible wavelength range at all incident angle due to the transition in the refractive index profile from air to the nanostructure layer that improved the anti-reflection characteristics.

Site of potential operating microscope light-induced phototoxicity on the human retina during temporal approach eye surgery.

PubMed

Pavilack, M A; Brod, R D

2001-02-01

To determine the site of focal illumination on the retina of phakic human cadaver eyes from an operating microscope positioned for temporal approach eye surgery. Experimental study. A Zeiss OPMI-6SFR operating microscope (Zeiss Humphrey Systems, Dublin, CA) was positioned over two phakic human cadaver eyes to measure the site of the focal illumination on the retina by directly observing the illumination on the posterior scleral surface of the globe. External localization of the foveola was made by direct observation using scleral indentation and indirect ophthalmoscopy. Various combinations of microscope angulation and field of view were analyzed. Distance of focal illumination from the operating room microscope relative to the foveola was measured. The diameter of the "hot spot" of focal illumination on the retina was 4.0 mm. With the eye positioned straight ahead and the level operating room microscope positioned for temporal approach eye surgery, the center of retinal illumination was 0.9 and 1.4 mm nasal relative to the foveola when the microscope field of view was centered over the cornea and temporal limbus, respectively. With the microscope angled 5, 10, 15, and 20 degrees temporally (oculars tilted toward surgeon), the center of the illumination was displaced nasal to the foveola by 1.1, 1.5, 3.8, and 5.1 mm, respectively, when the field of view was centered over the cornea and 1.5, 2.6, 4.7, and 6.0 mm, respectively, nasal to the foveola when centered over the temporal limbus. Retinal illumination from an operating microscope positioned for temporal approach eye surgery has the potential for light-induced injury to the fovea. Angulation of the operating microscope by up to 10 degrees temporally when the microscope field of view was centered over the cornea and up to 5 degrees temporally when centered over the temporal limbus was not adequate to displace the focal illumination off the foveola when the eye was in the straight-ahead position. Tilting the operating microscope 15 degrees or more temporally when centered on the pupil and 10 degrees or more when centered over the temporal limbus should safely displace the retinal light exposure away from the fovea during temporal approach surgery. Suggestions for reducing the risk of iatrogenic phototoxicity are reviewed.

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.

Compact Microscope Imaging System With Intelligent Controls Improved

NASA Technical Reports Server (NTRS)

McDowell, Mark

2004-01-01

The Compact Microscope Imaging System (CMIS) with intelligent controls is a diagnostic microscope analysis tool with intelligent controls for use in space, industrial, medical, and security applications. This compact miniature microscope, which can perform tasks usually reserved for conventional microscopes, has unique advantages in the fields of microscopy, biomedical research, inline process inspection, and space science. Its unique approach integrates a machine vision technique with an instrumentation and control technique that provides intelligence via the use of adaptive neural networks. The CMIS system was developed at the NASA Glenn Research Center specifically for interface detection used for colloid hard spheres experiments; biological cell detection for patch clamping, cell movement, and tracking; and detection of anode and cathode defects for laboratory samples using microscope technology.

Automated Diatom Analysis Applied to Traditional Light Microscopy: A Proof-of-Concept Study

NASA Astrophysics Data System (ADS)

Little, Z. H. L.; Bishop, I.; Spaulding, S. A.; Nelson, H.; Mahoney, C.

2017-12-01

Diatom identification and enumeration by high resolution light microscopy is required for many areas of research and water quality assessment. Such analyses, however, are both expertise and labor-intensive. These challenges motivate the need for an automated process to efficiently and accurately identify and enumerate diatoms. Improvements in particle analysis software have increased the likelihood that diatom enumeration can be automated. VisualSpreadsheet software provides a possible solution for automated particle analysis of high-resolution light microscope diatom images. We applied the software, independent of its complementary FlowCam hardware, to automated analysis of light microscope images containing diatoms. Through numerous trials, we arrived at threshold settings to correctly segment 67% of the total possible diatom valves and fragments from broad fields of view. (183 light microscope images were examined containing 255 diatom particles. Of the 255 diatom particles present, 216 diatoms valves and fragments of valves were processed, with 170 properly analyzed and focused upon by the software). Manual analysis of the images yielded 255 particles in 400 seconds, whereas the software yielded a total of 216 particles in 68 seconds, thus highlighting that the software has an approximate five-fold efficiency advantage in particle analysis time. As in past efforts, incomplete or incorrect recognition was found for images with multiple valves in contact or valves with little contrast. The software has potential to be an effective tool in assisting taxonomists with diatom enumeration by completing a large portion of analyses. Benefits and limitations of the approach are presented to allow for development of future work in image analysis and automated enumeration of traditional light microscope images containing diatoms.

Distribution of melanosomes across the retinal pigment epithelium of a hooded rat: implications for light damage

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

Howell, W.L.; Rapp, L.M.; Williams, T.P.

1982-02-01

Distribution of melanosomes across the retinal pigment epithelium of hooded rats (Long-Evans) is studied at the light microscopic and electron microscopic levels. This distribution is shown to be nonuniform: more melanosomes exist in the periphery than elsewhere and, importantly, there are very few melanosomes in a restricted area of the central portion of the superior hemisphere compared with the corresponding part of the inferior hemisphere. The region with fewest melanosomes is precisely the one that is highly susceptible to light damage. Because this region is the same in both pigmented and albino eyes, the paucity of melanin in this regionmore » is not the cause of its great sensitivity to light damage. Nor does light cause the nonuniform distribution of melanin. A possible explanation, involving a proposed vestigial tapetum, is given in order to explain the correlation of melanosome counts and sensitivity to light damage.« less

Calibrating excitation light fluxes for quantitative light microscopy in cell biology

PubMed Central

Grünwald, David; Shenoy, Shailesh M; Burke, Sean; Singer, Robert H

2011-01-01

Power output of light bulbs changes over time and the total energy delivered will depend on the optical beam path of the microscope, filter sets and objectives used, thus making comparison between experiments performed on different microscopes complicated. Using a thermocoupled power meter, it is possible to measure the exact amount of light applied to a specimen in fluorescence microscopy, regardless of the light source, as the light power measured can be translated into a power density at the sample. This widely used and simple tool forms the basis of a new degree of calibration precision and comparability of results among experiments and setups. Here we describe an easy-to-follow protocol that allows researchers to precisely estimate excitation intensities in the object plane, using commercially available opto-mechanical components. The total duration of this protocol for one objective and six filter cubes is 75 min including start-up time for the lamp. PMID:18974739

Development of an imaging system for single droplet characterization using a droplet generator.

PubMed

Minov, S Vulgarakis; Cointault, F; Vangeyte, J; Pieters, J G; Hijazi, B; Nuyttens, D

2012-01-01

The spray droplets generated by agricultural nozzles play an important role in the application accuracy and efficiency of plant protection products. The limitations of the non-imaging techniques and the recent improvements in digital image acquisition and processing increased the interest in using high speed imaging techniques in pesticide spray characterisation. The goal of this study was to develop an imaging technique to evaluate the characteristics of a single spray droplet using a piezoelectric single droplet generator and a high speed imaging technique. Tests were done with different camera settings, lenses, diffusers and light sources. The experiments have shown the necessity for having a good image acquisition and processing system. Image analysis results contributed in selecting the optimal set-up for measuring droplet size and velocity which consisted of a high speed camera with a 6 micros exposure time, a microscope lens at a working distance of 43 cm resulting in a field of view of 1.0 cm x 0.8 cm and a Xenon light source without diffuser used as a backlight. For measuring macro-spray characteristics as the droplet trajectory, the spray angle and the spray shape, a Macro Video Zoom lens at a working distance of 14.3 cm with a bigger field of view of 7.5 cm x 9.5 cm in combination with a halogen spotlight with a diffuser and the high speed camera can be used.

Hard X-Ray Scanning Microscope with Multilayer Laue Lens Nanofocusing Optics

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

Nazaretski, Evgeny

Evgeny Nazaretski, a physicist at Brookhaven Lab’s National Synchrotron Light Source II, spearheaded the development of a one-of-a-kind x-ray microscope with novel nanofocusing optics called multilayer Laue lenses.

Microscope-Based Fluid Physics Experiments in the Fluids and Combustion Facility on ISS

NASA Technical Reports Server (NTRS)

Doherty, Michael P.; Motil, Susan M.; Snead, John H.; Malarik, Diane C.

2000-01-01

At the NASA Glenn Research Center, the Microgravity Science Program is planning to conduct a large number of experiments on the International Space Station in both the Fluid Physics and Combustion Science disciplines, and is developing flight experiment hardware for use within the International Space Station's Fluids and Combustion Facility. Four fluids physics experiments that require an optical microscope will be sequentially conducted within a subrack payload to the Fluids Integrated Rack of the Fluids and Combustion Facility called the Light Microscopy Module, which will provide the containment, changeout, and diagnostic capabilities to perform the experiments. The Light Microscopy Module is planned as a fully remotely controllable on-orbit microscope facility, allowing flexible scheduling and control of experiments within International Space Station resources. This paper will focus on the four microscope-based experiments, specifically, their objectives and the sample cell and instrument hardware to accommodate their requirements.

Comparisons between conventional optical imaging and parametric indirect microscopic imaging on human skin detection

NASA Astrophysics Data System (ADS)

Liu, Guoyan; Gao, Kun; Liu, Xuefeng; Ni, Guoqiang

2016-10-01

We report a new method, polarization parameters indirect microscopic imaging with a high transmission infrared light source, to detect the morphology and component of human skin. A conventional reflection microscopic system is used as the basic optical system, into which a polarization-modulation mechanics is inserted and a high transmission infrared light source is utilized. The near-field structural characteristics of human skin can be delivered by infrared waves and material coupling. According to coupling and conduction physics, changes of the optical wave parameters can be calculated and curves of the intensity of the image can be obtained. By analyzing the near-field polarization parameters in nanoscale, we can finally get the inversion images of human skin. Compared with the conventional direct optical microscope, this method can break diffraction limit and achieve a super resolution of sub-100nm. Besides, the method is more sensitive to the edges, wrinkles, boundaries and impurity particles.

[Phenotype-based primary screening for drugs promoting neuronal subtype differentiation in embryonic stem cells with light microscope].

PubMed

Gao, Yi-ning; Wang, Dan-ying; Pan, Zong-fu; Mei, Yu-qin; Wang, Zhi-qiang; Zhu, Dan-yan; Lou, Yi-jia

2012-07-01

To set up a platform for phenotype-based primary screening of drug candidates promoting neuronal subtype differentiation in embryonic stem cells (ES) with light microscope. Hanging drop culture 4-/4+ method was employed to harvest the cells around embryoid body (EB) at differentiation endpoint. Morphological evaluation for neuron-like cells was performed with light microscope. Axons for more than three times of the length of the cell body were considered as neuron-like cells. The compound(s) that promote neuron-like cells was further evaluated. Icariin (ICA, 10(-6)mol/L) and Isobavachin (IBA, 10(-7)mol/L) were selected to screen the differentiation-promoting activity on ES cells. Immunofluorescence staining with specific antibodies (ChAT, GABA) was used to evaluate the neuron subtypes. The cells treated with IBA showed neuron-like phenotype, but the cells treated with ICA did not exhibit the morphological changes. ES cells treated with IBA was further confirmed to be cholinergic and GABAergic neurons. Phenotypic screening with light microscope for molecules promoting neuronal differentiation is an effective method with advantages of less labor and material consuming and time saving, and false-positive results derived from immunofluorescence can be avoided. The method confirms that IBA is able to facilitate ES cells differentiating into neuronal cells, including cholinergic neurons and GABAergic neurons.

From Animaculum to single molecules: 300 years of the light microscope

PubMed Central

Wollman, Adam J. M.; Nudd, Richard; Hedlund, Erik G.; Leake, Mark C.

2015-01-01

Although not laying claim to being the inventor of the light microscope, Antonj van Leeuwenhoek (1632–1723) was arguably the first person to bring this new technological wonder of the age properly to the attention of natural scientists interested in the study of living things (people we might now term ‘biologists’). He was a Dutch draper with no formal scientific training. From using magnifying glasses to observe threads in cloth, he went on to develop over 500 simple single lens microscopes (Baker & Leeuwenhoek 1739 Phil. Trans. 41, 503–519. (doi:10.1098/rstl.1739.0085)) which he used to observe many different biological samples. He communicated his finding to the Royal Society in a series of letters (Leeuwenhoek 1800 The select works of Antony Van Leeuwenhoek, containing his microscopical discoveries in many of the works of nature, vol. 1) including the one republished in this edition of Open Biology. Our review here begins with the work of van Leeuwenhoek before summarizing the key developments over the last ca 300 years, which has seen the light microscope evolve from a simple single lens device of van Leeuwenhoek's day into an instrument capable of observing the dynamics of single biological molecules inside living cells, and to tracking every cell nucleus in the development of whole embryos and plants. PMID:25924631

New Windows on the Biological World

ERIC Educational Resources Information Center

Arehart-Treichel, Joan

1975-01-01

Describes two new microscopes, the acoustic microscope and a scanning transmission microscope, both of which promise to yield fresh insights, based on revolutionary techniques into cellular biology. (BR)

Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy

NASA Astrophysics Data System (ADS)

Descloux, A.; Grußmayer, K. S.; Bostan, E.; Lukes, T.; Bouwens, A.; Sharipov, A.; Geissbuehler, S.; Mahul-Mellier, A.-L.; Lashuel, H. A.; Leutenegger, M.; Lasser, T.

2018-03-01

Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going `beyond the diffraction barrier' comes at a price, since most far-field super-resolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase imaging with fluorescence to provide high-speed imaging and spatial super-resolution. The non-iterative phase retrieval relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of eight planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument. The 4D microscope platform unifies the sensitivity and high temporal resolution of phase imaging with the specificity and high spatial resolution of fluorescence microscopy.

Synthesis, characterization, and application of novel biodegradable self-assembled 2-(N-phthalimido) ethyl-palmitate nanoparticles for cancer therapy

NASA Astrophysics Data System (ADS)

Kasoju, Naresh; Bora, Debajeet K.; Bhonde, Ramesh R.; Bora, Utpal

2010-03-01

We report the synthesis of novel biodegradable nanoparticles (NPs) which can kill the cancer cells without any additional drug loading. The NP was a self-assembled form of a phthalimide based conjugate, in which the phthalimide moiety was responsible for the anticancer activity. We describe the synthesis of a novel 2-(N-phthalimido) ethyl palmitate (PHEP-Pal) conjugate and subsequent preparation of NPs by a simple self assembly process. The successful synthesis of conjugate was confirmed by various characterization studies including nuclear magnetic resonance spectroscope, Fourier transform infrared spectroscope, TOF-liquid chromatography mass spectroscope, differential scanning calorimetry, and X-ray diffraction unit. The synthesis, shape, size, and size distribution of PHEP-Pal NPs were determined by transmission electron microscope, atomic force microscope, and dynamic light scattering technique. Finally, cell culture studies using A549 and HeLa cells were done to evaluate the anticancer effect of PHEP-Pal NPs, which demonstrated the potency of these NPs for use in cancer chemotherapy.

Red tea leaves infusion as a reducing and stabilizing agent in silver nanoparticles synthesis

NASA Astrophysics Data System (ADS)

Pluta, K.; Tryba, A. M.; Malina, D.; Sobczak-Kupiec, A.

2017-12-01

Due to the unique properties of silver nanoparticles there is growing interest in their applications. Current trends in nanotechnology are focused on developing a new technique to synthesize nanoparticles using biological methods associated with the use of plant extracts, fungi, bacteria or essential oils. These methods are a promising alternative to conventional approaches which can minimize the use of hazardous substances. The silver nanoparticles synthesis using red tea infusion as a reducing and stabilizing agent and their characteristics have been described. Total antioxidant capacity using DPPH radical and total content of phenolic compounds by Folin-Ciocalteau method were measured in tea infusion. Synthesis of silver nanoparticles was carried out using chemical reduction at various temperatures. Furthermore, the effect of tea infusion volume added to reaction mixture on nanoparticles’ properties was investigated. Finally, nanosilver suspensions were characterized by UV-vis spectrophotometer, dynamic light scattering (DLS) scanning electron microscope (SEM) and transmission electron microscope (TEM). Moreover, phytotoxicity of silver nanoparticles was determined using Phytotestkit microbiotest.

Chip-based wide field-of-view nanoscopy

NASA Astrophysics Data System (ADS)

Diekmann, Robin; Helle, Øystein I.; Øie, Cristina I.; McCourt, Peter; Huser, Thomas R.; Schüttpelz, Mark; Ahluwalia, Balpreet S.

2017-04-01

Present optical nanoscopy techniques use a complex microscope for imaging and a simple glass slide to hold the sample. Here, we demonstrate the inverse: the use of a complex, but mass-producible optical chip, which hosts the sample and provides a waveguide for the illumination source, and a standard low-cost microscope to acquire super-resolved images via two different approaches. Waveguides composed of a material with high refractive-index contrast provide a strong evanescent field that is used for single-molecule switching and fluorescence excitation, thus enabling chip-based single-molecule localization microscopy. Additionally, multimode interference patterns induce spatial fluorescence intensity variations that enable fluctuation-based super-resolution imaging. As chip-based nanoscopy separates the illumination and detection light paths, total-internal-reflection fluorescence excitation is possible over a large field of view, with up to 0.5 mm × 0.5 mm being demonstrated. Using multicolour chip-based nanoscopy, we visualize fenestrations in liver sinusoidal endothelial cells.

Toward Microscopic Equations of State for Core-Collapse Supernovae from Chiral Effective Field Theory

NASA Astrophysics Data System (ADS)

Aboona, Bassam; Holt, Jeremy

2017-09-01

Chiral effective field theory provides a modern framework for understanding the structure and dynamics of nuclear many-body systems. Recent works have had much success in applying the theory to describe the ground- and excited-state properties of light and medium-mass atomic nuclei when combined with ab initio numerical techniques. Our aim is to extend the application of chiral effective field theory to describe the nuclear equation of state required for supercomputer simulations of core-collapse supernovae. Given the large range of densities, temperatures, and proton fractions probed during stellar core collapse, microscopic calculations of the equation of state require large computational resources on the order of one million CPU hours. We investigate the use of graphics processing units (GPUs) to significantly reduce the computational cost of these calculations, which will enable a more accurate and precise description of this important input to numerical astrophysical simulations. Cyclotron Institute at Texas A&M, NSF Grant: PHY 1659847, DOE Grant: DE-FG02-93ER40773.

Endoscopic vs. microscopic transsphenoidal surgery for Cushing's disease: a systematic review and meta-analysis.

PubMed

Broersen, Leonie H A; Biermasz, Nienke R; van Furth, Wouter R; de Vries, Friso; Verstegen, Marco J T; Dekkers, Olaf M; Pereira, Alberto M

2018-05-16

Systematic review and meta-analysis comparing endoscopic and microscopic transsphenoidal surgery for Cushing's disease regarding surgical outcomes (remission, recurrence, and mortality) and complication rates. To stratify the results by tumor size. Nine electronic databases were searched in February 2017 to identify potentially relevant articles. Cohort studies assessing surgical outcomes or complication rates after endoscopic or microscopic transsphenoidal surgery for Cushing's disease were eligible. Pooled proportions were reported including 95% confidence intervals. We included 97 articles with 6695 patients in total (5711 microscopically and 984 endoscopically operated). Overall, remission was achieved in 5177 patients (80%), with no clear difference between both techniques. Recurrence was around 10% and short term mortality < 0.5% for both techniques. Cerebrospinal fluid leak occurred more often in endoscopic surgery (12.9 vs. 4.0%), whereas transient diabetes insipidus occurred less often (11.3 vs. 21.7%). For microadenomas, results were comparable between both techniques. For macroadenomas, the percentage of patients in remission was higher after endoscopic surgery (76.3 vs. 59.9%), and the percentage recurrence lower after endoscopic surgery (1.5 vs. 17.0%). Endoscopic surgery for patients with Cushing's disease reaches comparable results for microadenomas, and probably better results for macroadenomas than microscopic surgery. This is present despite the presumed learning curve of the newer endoscopic technique, although confounding cannot be excluded. Based on this study, endoscopic surgery may thus be considered the current standard of care. Microscopic surgery can be used based on neurosurgeon's preference. Endocrinologists and neurosurgeons in pituitary centers performing the microscopic technique should at least consider referring Cushing's disease patients with a macroadenoma.

Evaluation of mobile digital light-emitting diode fluorescence microscopy in Hanoi, Viet Nam.

PubMed

Chaisson, L H; Reber, C; Phan, H; Switz, N; Nilsson, L M; Myers, F; Nhung, N V; Luu, L; Pham, T; Vu, C; Nguyen, H; Nguyen, A; Dinh, T; Nahid, P; Fletcher, D A; Cattamanchi, A

2015-09-01

Hanoi Lung Hospital, Hanoi, Viet Nam. To compare the accuracy of CellScopeTB, a manually operated mobile digital fluorescence microscope, with conventional microscopy techniques. Patients referred for sputum smear microscopy to the Hanoi Lung Hospital from May to September 2013 were included. Ziehl-Neelsen (ZN) smear microscopy, conventional light-emitting diode (LED) fluorescence microscopy (FM), CellScopeTB-based LED FM and Xpert(®) MTB/RIF were performed on sputum samples. The sensitivity and specificity of microscopy techniques were determined in reference to Xpert results, and differences were compared using McNemar's paired test of proportions. Of 326 patients enrolled, 93 (28.5%) were Xpert-positive for TB. The sensitivity of ZN microscopy, conventional LED FM, and CellScopeTB-based LED FM was respectively 37.6% (95%CI 27.8-48.3), 41.9% (95%CI 31.8-52.6), and 35.5% (95%CI 25.8-46.1). The sensitivity of CellScopeTB was similar to that of conventional LED FM (difference -6.5%, 95%CI -18.2 to 5.3, P = 0.33) and ZN microscopy (difference -2.2%, 95%CI -9.2 to 4.9, P = 0.73). The specificity was >99% for all three techniques. CellScopeTB performed similarly to conventional microscopy techniques in the hands of experienced TB microscopists. However, the sensitivity of all sputum microscopy techniques was low. Options enabled by digital microscopy, such as automated imaging with real-time computerized analysis, should be explored to increase sensitivity.

Cornea and ocular lens visualized with three-dimensional confocal microscopy

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1992-08-01

This paper demonstrates the advantages of three-dimensional reconstruction of the cornea and the ocular crystalline lens by confocal microscopy and volume rendering computer techniques. The advantages of noninvasive observation of ocular structures in living, unstained, unfixed tissue include the following: the tissue is in a natural living state without the artifacts of fixation, mechanical sectioning, and staining; the three-dimensional structure can be observed from any view point and quantitatively analyzed; the dynamics of morphological changes can be studied; and the use of confocal microscopic observation results in a reduction of the number of animals required for ocular morphometric studies. The main advantage is that the dynamic morphology of ocular structures can be investigated in living ocular tissue. A laser scanning confocal microscope was used in the reflected light mode to obtain the two- dimensional images from the cornea and the ocular lens of a freshly enucleated rabbit eye. The light source was an argon ion laser with 488 nm wavelength. The microscope objective was a Leitz 25X, NA 0.6 water immersion lens. The 400 micron thick cornea was optically sectioned into 133, three micron sections. The semi-transparent cornea and the in-situ ocular lens was visualized as high resolution, high contrast two-dimensional images. The under sampling resulted in a three-dimensional visualization rendering in which the corneal thickness (z-axis) is compressed. The structures observed in the cornea include: superficial epithelial cells and their nuclei, basal epithelial cells and their `beaded' cell borders, basal lamina, nerve plexus, nerve fibers, free nerve endings in the basal epithelial cells, nuclei of stromal keratocytes, and endothelial cells. The structures observed in the in-situ ocular lens include: lens capsule, lens epithelial cells, and individual lens fibers.

Backscattered electron SEM imaging of resin sections from plant specimens: observation of histological to subcellular structure and CLEM.

PubMed

Rizzo, N W; Duncan, K E; Bourett, T M; Howard, R J

2016-08-01

We have refined methods for biological specimen preparation and low-voltage backscattered electron imaging in the scanning electron microscope that allow for observation at continuous magnifications of ca. 130-70 000 X, and documentation of tissue and subcellular ultrastructure detail. The technique, based upon early work by Ogura & Hasegawa (1980), affords use of significantly larger sections from fixed and resin-embedded specimens than is possible with transmission electron microscopy while providing similar data. After microtomy, the sections, typically ca. 750 nm thick, were dried onto the surface of glass or silicon wafer and stained with heavy metals-the use of grids avoided. The glass/wafer support was then mounted onto standard scanning electron microscopy sample stubs, carbon-coated and imaged directly at an accelerating voltage of 5 kV, using either a yttrium aluminum garnet or ExB backscattered electron detector. Alternatively, the sections could be viewed first by light microscopy, for example to document signal from a fluorescent protein, and then by scanning electron microscopy to provide correlative light/electron microscope (CLEM) data. These methods provide unobstructed access to ultrastructure in the spatial context of a section ca. 7 × 10 mm in size, significantly larger than the typical 0.2 × 0.3 mm section used for conventional transmission electron microscopy imaging. Application of this approach was especially useful when the biology of interest was rare or difficult to find, e.g. a particular cell type, developmental stage, large organ, the interface between cells of interacting organisms, when contextual information within a large tissue was obligatory, or combinations of these factors. In addition, the methods were easily adapted for immunolocalizations. © 2015 The Author. Journal of Microscopy published by John Wiley & Sons, Ltd on behalf of the Royal Microscopical Society.

Surface imaging microscope

NASA Astrophysics Data System (ADS)

Rogala, Eric W.; Bankman, Isaac N.

2008-04-01

The three-dimensional shapes of microscopic objects are becoming increasingly important for battlespace CBRNE sensing. Potential applications of microscopic 3D shape observations include characterization of biological weapon particles and manufacturing of micromechanical components. Aerosol signatures of stand-off lidar systems, using elastic backscatter or polarization, are dictated by the aerosol particle shapes and sizes that must be well characterized in the lab. A low-cost, fast instrument for 3D surface shape microscopy will be a valuable point sensor for biological particle sensing applications. Both the cost and imaging durations of traditional techniques such as confocal microscopes, atomic force microscopes, and electron scanning microscopes are too high. We investigated the feasibility of a low-cost, fast interferometric technique for imaging the 3D surface shape of microscopic objects at frame rates limited only by the camera in the system. The system operates at two laser wavelengths producing two fringe images collected simultaneously by a digital camera, and a specialized algorithm we developed reconstructs the surface map of the microscopic object. The current implementation assembled to test the concept and develop the new 3D reconstruction algorithm has 0.25 micron resolution in the x and y directions, and about 0.1 micron accuracy in the z direction, as tested on a microscopic glass test object manufactured with etching techniques. We describe the interferometric instrument, present the reconstruction algorithm, and discuss further development.

Genetically expressed voltage sensor ArcLight for imaging large scale cortical activity in the anesthetized and awake mouse

PubMed Central

Borden, Peter Y.; Ortiz, Alex D.; Waiblinger, Christian; Sederberg, Audrey J.; Morrissette, Arthur E.; Forest, Craig R.; Jaeger, Dieter; Stanley, Garrett B.

2017-01-01

Abstract. With the recent breakthrough in genetically expressed voltage indicators (GEVIs), there has been a tremendous demand to determine the capabilities of these sensors in vivo. Novel voltage sensitive fluorescent proteins allow for direct measurement of neuron membrane potential changes through changes in fluorescence. Here, we utilized ArcLight, a recently developed GEVI, and examined the functional characteristics in the widely used mouse somatosensory whisker pathway. We measured the resulting evoked fluorescence using a wide-field microscope and a CCD camera at 200 Hz, which enabled voltage recordings over the entire cortical region with high temporal resolution. We found that ArcLight produced a fluorescent response in the S1 barrel cortex during sensory stimulation at single whisker resolution. During wide-field cortical imaging, we encountered substantial hemodynamic noise that required additional post hoc processing through noise subtraction techniques. Over a period of 28 days, we found clear and consistent ArcLight fluorescence responses to a simple sensory input. Finally, we demonstrated the use of ArcLight to resolve cortical S1 sensory responses in the awake mouse. Taken together, our results demonstrate the feasibility of ArcLight as a measurement tool for mesoscopic, chronic imaging. PMID:28491905

Biophysical characterization of genistein-membrane interaction and its correlation with biological effect on cells - The case of EYPC liposomes and human erythrocyte membranes.

PubMed

Pawlikowska-Pawlęga, Bożena; Misiak, Lucjan E; Jarosz-Wilkołazka, Anna; Zarzyka, Barbara; Paduch, Roman; Gawron, Antoni; Gruszecki, Wieslaw I

2014-08-01

With application of EPR and (1)H NMR techniques genistein interaction with liposomes formed with egg yolk lecithin and with erythrocyte membranes was assessed. The present study addressed the problem of genistein localization and its effects on lipid membrane fluidity and protein conformation. The range of microscopic techniques was employed to study genistein effects on HeLa cells and human erythrocytes. Moreover, DPPH bioassay, superoxide anion radical test and enzymatic measurements were performed in HeLa cells subjected to genistein. The gathered results from both EPR and NMR techniques indicated strong ordering effect of genistein on the motional freedom of lipids in the head group region and the adjacent hydrophobic zone in liposomal as well as in red blood cell membranes. EPR study of human ghost showed also the changes in the erythrocyte membrane protein conformation. The membrane effects of genistein were correlated with the changes in internal membranes arrangement of HeLa cells as it was noticed using transmission electron microscopic and fluorescent techniques. Scanning electron and light microscopy methods showed that one of the aftermaths of genistein incorporation into membranes was creation of echinocytic form of the red blood cells with reduced diameter. Genistein improved redox status of HeLa cells treated with H2O2 by lowering radicals' level. In conclusion, the capacity of genistein to incorporate, to affect membrane organization and to change its biophysical properties is correlated with the changes inside the cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparative cost assessment of the Kato-Katz and FLOTAC techniques for soil-transmitted helminth diagnosis in epidemiological surveys

PubMed Central

2010-01-01

Background The Kato-Katz technique is widely used for the diagnosis of soil-transmitted helminthiasis in epidemiological surveys and is believed to be an inexpensive method. The FLOTAC technique shows a higher sensitivity for the diagnosis of light-intensity soil-transmitted helminth infections but is reported to be more complex and expensive. We assessed the costs related to the collection, processing and microscopic examination of stool samples using the Kato-Katz and FLOTAC techniques in an epidemiological survey carried out in Zanzibar, Tanzania. Methods We measured the time for the collection of a single stool specimen in the field, transfer to a laboratory, preparation and microscopic examination using standard protocols for the Kato-Katz and FLOTAC techniques. Salaries of health workers, life expectancy and asset costs of materials, and infrastructure costs were determined. The average cost for a single or duplicate Kato-Katz thick smears and the FLOTAC dual or double technique were calculated. Results The average time needed to collect a stool specimen and perform a single or duplicate Kato-Katz thick smears or the FLOTAC dual or double technique was 20 min and 34 sec (20:34 min), 27:21 min, 28:14 min and 36:44 min, respectively. The total costs for a single and duplicate Kato-Katz thick smears were US$ 1.73 and US$ 2.06, respectively, and for the FLOTAC double and dual technique US$ 2.35 and US$ 2.83, respectively. Salaries impacted most on the total costs of either method. Conclusions The time and cost for soil-transmitted helminth diagnosis using either the Kato-Katz or FLOTAC method in epidemiological surveys are considerable. Our results can help to guide healthcare decision makers and scientists in budget planning and funding for epidemiological surveys, anthelminthic drug efficacy trials and monitoring of control interventions. PMID:20707931

The influence of filling technique on depth of tubule penetration by root canal sealer: a study using light microscopy and digital image processing.

PubMed

De Deus, Gustavo A; Gurgel-Filho, Eduardo Diogo; Maniglia-Ferreira, Cláudio; Coutinho-Filho, Tauby

2004-04-01

The purpose of this study was to compare the depth of sealer penetration into dentinal tubules by three root-filling techniques using light microscopy and digital image processing. Thirty-two maxillary central incisors were prepared. Two teeth were separated for the control group. The rest were divided into three equal groups and obturated as following--G1: lateral condensation; G2: warm vertical compaction of gutta-percha and G3: Thermafil system. Each sample was sectioned longitudinally and prepared for microscopic analysis. A sequence of photomicrographs with magnifications of X50, X200 and X500 were taken. Through digital image analysis and processing, measurements for each field were obtained. A non-parametric ANOVA Kruskal-Wallis analysis was used to determine whether there were significant differences among the groups. Significant differences between G2 and G1 (p = 0.034) and between G3 and G1 (p = 0.021) were identified. There were no significant differences between G2 and G3 (p > 0.05). The results of this research suggest that samples root-filled by thermoplasticised gutta-percha techniques lead to deeper penetration of the root canal sealer into the dentinal tubules.

Optical detection of ultrasound using an apertureless near-field scanning optical microscopy system

NASA Astrophysics Data System (ADS)

Ahn, Phillip; Zhang, Zhen; Sun, Cheng; Balogun, Oluwaseyi

2013-01-01

Laser ultrasonics techniques are power approaches for non-contact generation and detection of high frequency ultrasound on a local scale. In these techniques, optical diffraction limits the spatial information that can be accessed from a measurement. In order to improve the lateral spatial resolution, we incorporate an apertureless near-field scanning optical microscope (aNSOM) into laser ultrasonics setup for local detection of laser generated ultrasound. The aNSOM technique relies on the measurement of a weak backscattered near-field light intensity resulting from the oblique illumination of a nanoscale probe-tip positioned close to a sample surface. We enhance the optical near-field intensity by coupling light to surface plasmon polaritons (SPPs) on the shaft of an atomic force microscopy (AFM) cantilever. The SPPs propagate down the AFM shaft, localize at the tip apex, and are backscattered to the far-field when the separation distance between the probe tip and the sample surface is comparable to the probe-tip radius. The backscattered near-field intensity is dynamically modulated when an ultrasonic wave arrives at the sample surface leading to a transient change in the tip-sample separation distance. We present experimental results detailing measurement of broadband and narrowband laser generated ultrasound in solids with frequencies reaching up to 180 MHz range.

Microscopic video observation of capillary vessel systems using diffuse back lighting

NASA Astrophysics Data System (ADS)

Sakai, Minako; Arai, Hiroki; Iwai, Toshiaki

2017-04-01

We have been developing a simple and practical video microscopy system based on absorption spectra of biological substance to perform spectroscopic observation of living tissues. The diffuse backlighting effect is actively used in the developed system, which is generated by multiple light scattering in the tissue. It is demonstrated that the light specularly reflected from the skin surface can be completely suppressed in the microscopic observation and the biological activity of the capillary vessel systems distributed under the skin can be successfully observed. As a result, we can confirm the effectiveness of the video microscopy system using diffuse backlighting and the applicability of our developed system.

Use of digital micromirror devices as dynamic pinhole arrays for adaptive confocal fluorescence microscopy

NASA Astrophysics Data System (ADS)

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

2018-02-01

In this work, we present a new confocal laser scanning microscope capable to perform sensorless wavefront optimization in real time. The device is a parallelized laser scanning microscope in which the excitation light is structured in a lattice of spots by a spatial light modulator, while a deformable mirror provides aberration correction and scanning. A binary DMD is positioned in an image plane of the detection optical path, acting as a dynamic array of reflective confocal pinholes, images by a high performance cmos camera. A second camera detects images of the light rejected by the pinholes for sensorless aberration correction.

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.

Coherent anti-Stokes Raman scattering spectroscope/microscope based on a widely tunable laser source

NASA Astrophysics Data System (ADS)

Dementjev, A.; Gulbinas, V.; Serbenta, A.; Kaucikas, M.; Niaura, G.

2010-03-01

We present a coherent anti-Stokes Raman scattering (CARS) microscope based on a robust and simple laser source. A picosecond laser operating in a cavity dumping regime at the 1 MHz repetition rate was used to pump a traveling wave optical parametric generator, which serves as a two-color excitation light source for the CARS microscope. We demonstrate the ability of the presented CARS microscope to measure CARS spectra and images by using several detection schemes.

Hard X-Ray Scanning Microscope with Multilayer Laue Lens Nanofocusing Optics

ScienceCinema

Nazaretski, Evgeny

2018-06-13

Evgeny Nazaretski, a physicist at Brookhaven Lab’s National Synchrotron Light Source II, spearheaded the development of a one-of-a-kind x-ray microscope with novel nanofocusing optics called multilayer Laue lenses.

Smart textile framework: Photochromic and fluorescent cellulosic fabric printed by strontium aluminate pigment.

PubMed

Khattab, Tawfik A; Rehan, Mohamed; Hamouda, Tamer

2018-09-01

Smart clothing can be defined as textiles that respond to a certain stimulus accompanied by a change in their properties. A specific class herein is the photochromic and fluorescent textiles that change color with light. A photochromic and fluorescent cotton fabric based on pigment printing is obtained. Such fabric is prepared by aqueous-based pigment-binder printing formulation containing inorganic pigment phosphor characterized by good photo- and thermal stability. It exhibits optimal excitation wavelength (365 nm) results in color and fluorescence change of the fabric surface. To prepare the transparent pigment-binder composite film, the phosphor pigment must be well-dispersed via physical immobilization without their aggregation. The pigment-binder paste is applied successfully onto cotton fabric using screen printing technique followed by thermal fixation. After screen-printing, a homogenous photochromic film is assembled on a cotton substrate surface, which represents substantial greenish-yellow color development as indicated by CIE Lab color space measurements under ultraviolet light, even at a pigment concentration of 0.08 wt% of the printing paste. The photochromic cotton fabric exhibit three excitation peaks at 272, 325 and 365 nm and three emission peaks at 418, 495 and 520 nm. The fluorescent optical microscope, scanning electron microscope, elemental mapping, energy dispersive X-ray spectroscopy, fluorescence emission and UV/Vis absorption spectroscopic data of the printed cotton fabric are described. The printed fabric showed a reversible and rapid photochromic response during ultra-violet excitation without fatigue. The fastness properties including washing, crocking, perspiration, sublimation/heat, and light are described. Copyright © 2018 Elsevier Ltd. All rights reserved.

Infrared spectroscopy of molecular submonolayers on surfaces by infrared scanning tunneling microscopy: tetramantane on Au111.

PubMed

Pechenezhskiy, Ivan V; Hong, Xiaoping; Nguyen, Giang D; Dahl, Jeremy E P; Carlson, Robert M K; Wang, Feng; Crommie, Michael F

2013-09-20

We have developed a new scanning-tunneling-microscopy-based spectroscopy technique to characterize infrared (IR) absorption of submonolayers of molecules on conducting crystals. The technique employs a scanning tunneling microscope as a precise detector to measure the expansion of a molecule-decorated crystal that is irradiated by IR light from a tunable laser source. Using this technique, we obtain the IR absorption spectra of [121]tetramantane and [123]tetramantane on Au(111). Significant differences between the IR spectra for these two isomers show the power of this new technique to differentiate chemical structures even when single-molecule-resolved scanning tunneling microscopy (STM) images look quite similar. Furthermore, the new technique was found to yield significantly better spectral resolution than STM-based inelastic electron tunneling spectroscopy, and to allow determination of optical absorption cross sections. Compared to IR spectroscopy of bulk tetramantane powders, infrared scanning tunneling microscopy (IRSTM) spectra reveal narrower and blueshifted vibrational peaks for an ordered tetramantane adlayer. Differences between bulk and surface tetramantane vibrational spectra are explained via molecule-molecule interactions.

Comparison of quartz crystallographic preferred orientations identified with optical fabric analysis, electron backscatter and neutron diffraction techniques.

PubMed

Hunter, N J R; Wilson, C J L; Luzin, V

2017-02-01

Three techniques are used to measure crystallographic preferred orientations (CPO) in a naturally deformed quartz mylonite: transmitted light cross-polarized microscopy using an automated fabric analyser, electron backscatter diffraction (EBSD) and neutron diffraction. Pole figure densities attributable to crystal-plastic deformation are variably recognizable across the techniques, particularly between fabric analyser and diffraction instruments. Although fabric analyser techniques offer rapid acquisition with minimal sample preparation, difficulties may exist when gathering orientation data parallel with the incident beam. Overall, we have found that EBSD and fabric analyser techniques are best suited for studying CPO distributions at the grain scale, where individual orientations can be linked to their source grain or nearest neighbours. Neutron diffraction serves as the best qualitative and quantitative means of estimating the bulk CPO, due to its three-dimensional data acquisition, greater sample area coverage, and larger sample size. However, a number of sampling methods can be applied to FA and EBSD data to make similar approximations. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Fabrication, characterization, and photocatalytic property of α-Fe2O3/graphene oxide composite

NASA Astrophysics Data System (ADS)

Li, Hong; Zhao, Qidong; Li, Xinyong; Zhu, Zhengru; Tade, Moses; Liu, Shaomin

2013-06-01

Spindle-shaped microstructure of α-Fe2O3 was successfully synthesized by a simple hydrothermal method. The α-Fe2O3/graphene oxide (GO) composites was prepared using a modified Hummers' strategy. The properties of the samples were systematically investigated by X-ray powder diffraction (XRD), UV-Vis diffuse reflectance spectrophotometer, transmission electron microscope, atomic force microscope, X-ray photoelectron spectroscopy, and Raman spectroscopy (Raman) techniques. GO nanosheets act as supporting materials for anchoring the α-Fe2O3 particles. The average crystallite sizes of the α-Fe2O3 and α-Fe2O3/GO samples are ca. 27 and 24 nm, respectively. The possible growth of α-Fe2O3 onto GO layers led to a higher absorbance capacity for visible light by α-Fe2O3/GO than α-Fe2O3 composite. The photocatalytic degradation of toluene over the α-Fe2O3 and α-Fe2O3/GO samples under xenon-lamp irradiation was comparatively studied by in situ FTIR technique. The results indicate that the α-Fe2O3/GO sample synthesized exhibited a higher capacity for the degradation of toluene. The composite of α-Fe2O3/GO could be promisingly applied in photo-driven air purification.

Automated imaging of cellular spheroids with selective plane illumination microscopy on a chip (Conference Presentation)

NASA Astrophysics Data System (ADS)

Paiè, Petra; Bassi, Andrea; Bragheri, Francesca; Osellame, Roberto

2017-02-01

Selective plane illumination microscopy (SPIM) is an optical sectioning technique that allows imaging of biological samples at high spatio-temporal resolution. Standard SPIM devices require dedicated set-ups, complex sample preparation and accurate system alignment, thus limiting the automation of the technique, its accessibility and throughput. We present a millimeter-scaled optofluidic device that incorporates selective plane illumination and fully automatic sample delivery and scanning. To this end an integrated cylindrical lens and a three-dimensional fluidic network were fabricated by femtosecond laser micromachining into a single glass chip. This device can upgrade any standard fluorescence microscope to a SPIM system. We used SPIM on a CHIP to automatically scan biological samples under a conventional microscope, without the need of any motorized stage: tissue spheroids expressing fluorescent proteins were flowed in the microchannel at constant speed and their sections were acquired while passing through the light sheet. We demonstrate high-throughput imaging of the entire sample volume (with a rate of 30 samples/min), segmentation and quantification in thick (100-300 μm diameter) cellular spheroids. This optofluidic device gives access to SPIM analyses to non-expert end-users, opening the way to automatic and fast screening of a high number of samples at subcellular resolution.

Quantitative locomotion study of freely swimming micro-organisms using laser diffraction.

PubMed

Magnes, Jenny; Susman, Kathleen; Eells, Rebecca

2012-10-25

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in contrast, have been conducted using microscope-based approaches, which limit the movement and behavior to a narrow, nearly two-dimensional focal field.(1) We present a novel analytical approach that provides real-time analysis of freely swimming C. elegans in a cuvette without dependence on microscope-based equipment. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through the cuvette. We measure oscillation frequencies for freely swimming nematodes. Analysis of the far-field diffraction patterns reveals clues about the waveforms of the nematodes. Diffraction is the process of light bending around an object. In this case light is diffracted by the organisms. The light waves interfere and can form a diffraction pattern. A far-field, or Fraunhofer, diffraction pattern is formed if the screen-to-object distance is much larger than the diffracting object. In this case, the diffraction pattern can be calculated (modeled) using a Fourier transform.(2) C. elegans are free-living soil-dwelling nematodes that navigate in three dimensions. They move both on a solid matrix like soil or agar in a sinusoidal locomotory pattern called crawling and in liquid in a different pattern called swimming.(3) The roles played by sensory information provided by mechanosensory, chemosensory, and thermosensory cells that govern plastic changes in locomotory patterns and switches in patterns are only beginning to be elucidated.(4) We describe an optical approach to measuring nematode locomotion in three dimensions that does not require a microscope and will enable us to begin to explore the complexities of nematode locomotion under different conditions.

Phase-shifting interference microscope with extendable field of measurement

NASA Astrophysics Data System (ADS)

Lin, Shyh-Tsong; Hsu, Wei-Feng; Wang, Ming-Shiang

2018-04-01

An innovative phase-shifting interference microscope aimed at extending the field of measurement is proposed in this paper. The microscope comprises a light source module, a phase modulation module, and an interferometric module, which reconstructs the micro-structure contours of samples using the five-step phase-shifting algorithm. This paper discusses the measurement theory and outlines the configuration, experimental setup, and experimental results obtained using the proposed interference microscope. The results confirm the efficacy of the microscope, achieving a standard deviation of 2.4 nm from a step height of 86.2 nm in multiple examinations.

Shaping the light for the investigation of depth-extended scattering media

NASA Astrophysics Data System (ADS)

Osten, W.; Frenner, K.; Pedrini, G.; Singh, A. K.; Schindler, J.; Takeda, M.

2018-02-01

Scattering media are an ongoing challenge for all kind of imaging technologies including coherent and incoherent principles. Inspired by new approaches of computational imaging and supported by the availability of powerful computers, spatial light modulators, light sources and detectors, a variety of new methods ranging from holography to time-of-flight imaging, phase conjugation, phase recovery using iterative algorithms and correlation techniques have been introduced and applied to different types of objects. However, considering the obvious progress in this field, several problems are still matter of investigation and their solution could open new doors for the inspection and application of scattering media as well. In particular, these open questions include the possibility of extending the 2d-approach to the inspection of depth-extended objects, the direct use of a scattering media as a simple tool for imaging of complex objects and the improvement of coherent inspection techniques for the dimensional characterization of incoherently radiating spots embedded in scattering media. In this paper we show our recent findings in coping with these challenges. First we describe how to explore depth-extended objects by means of a scattering media. Afterwards, we extend this approach by implementing a new type of microscope making use of a simple scatter plate as a kind of flat and unconventional imaging lens. Finally, we introduce our shearing interferometer in combination with structured illumination for retrieving the axial position of fluorescent light emitting spots embedded in scattering media.

The Light Microscopy Module: An On-Orbit Multi-User Microscope Facility

NASA Technical Reports Server (NTRS)

Motil, Susan M.; Snead, John H.

2002-01-01

The Light Microscopy Module (LMM) is planned as a remotely controllable on-orbit microscope subrack facility, allowing flexible scheduling and operation of fluids and biology experiments within the Fluids and Combustion Facility (FCF) Fluids Integrated Rack (FIR) on the International Space Station (ISS). The LMM will be the first integrated payload with the FIR to conduct four fluid physics experiments. A description of the LMM diagnostic capabilities, including video microscopy, interferometry, laser tweezers, confocal, and spectrophotometry, will be provided.

Ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation.

PubMed

Werley, Christopher A; Chien, Miao-Ping; Cohen, Adam E

2017-12-01

The rapid increase in the number and quality of fluorescent reporters and optogenetic actuators has yielded a powerful set of tools for recording and controlling cellular state and function. To achieve the full benefit of these tools requires improved optical systems with high light collection efficiency, high spatial and temporal resolution, and patterned optical stimulation, in a wide field of view (FOV). Here we describe our 'Firefly' microscope, which achieves these goals in a Ø6 mm FOV. The Firefly optical system is optimized for simultaneous photostimulation and fluorescence imaging in cultured cells. All but one of the optical elements are commercially available, yet the microscope achieves 10-fold higher light collection efficiency at its design magnification than the comparable commercially available microscope using the same objective. The Firefly microscope enables all-optical electrophysiology ('Optopatch') in cultured neurons with a throughput and information content unmatched by other neuronal phenotyping systems. This capability opens possibilities in disease modeling and phenotypic drug screening. We also demonstrate applications of the system to voltage and calcium recordings in human induced pluripotent stem cell derived cardiomyocytes.

Ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation

PubMed Central

Werley, Christopher A.; Chien, Miao-Ping; Cohen, Adam E.

2017-01-01

The rapid increase in the number and quality of fluorescent reporters and optogenetic actuators has yielded a powerful set of tools for recording and controlling cellular state and function. To achieve the full benefit of these tools requires improved optical systems with high light collection efficiency, high spatial and temporal resolution, and patterned optical stimulation, in a wide field of view (FOV). Here we describe our ‘Firefly’ microscope, which achieves these goals in a Ø6 mm FOV. The Firefly optical system is optimized for simultaneous photostimulation and fluorescence imaging in cultured cells. All but one of the optical elements are commercially available, yet the microscope achieves 10-fold higher light collection efficiency at its design magnification than the comparable commercially available microscope using the same objective. The Firefly microscope enables all-optical electrophysiology (‘Optopatch’) in cultured neurons with a throughput and information content unmatched by other neuronal phenotyping systems. This capability opens possibilities in disease modeling and phenotypic drug screening. We also demonstrate applications of the system to voltage and calcium recordings in human induced pluripotent stem cell derived cardiomyocytes. PMID:29296505

Holographic pixel super-resolution in portable lensless on-chip microscopy using a fiber-optic array.

PubMed

Bishara, Waheb; Sikora, Uzair; Mudanyali, Onur; Su, Ting-Wei; Yaglidere, Oguzhan; Luckhart, Shirley; Ozcan, Aydogan

2011-04-07

We report a portable lensless on-chip microscope that can achieve <1 µm resolution over a wide field-of-view of ∼ 24 mm(2) without the use of any mechanical scanning. This compact on-chip microscope weighs ∼ 95 g and is based on partially coherent digital in-line holography. Multiple fiber-optic waveguides are butt-coupled to light emitting diodes, which are controlled by a low-cost micro-controller to sequentially illuminate the sample. The resulting lensfree holograms are then captured by a digital sensor-array and are rapidly processed using a pixel super-resolution algorithm to generate much higher resolution holographic images (both phase and amplitude) of the objects. This wide-field and high-resolution on-chip microscope, being compact and light-weight, would be important for global health problems such as diagnosis of infectious diseases in remote locations. Toward this end, we validate the performance of this field-portable microscope by imaging human malaria parasites (Plasmodium falciparum) in thin blood smears. Our results constitute the first-time that a lensfree on-chip microscope has successfully imaged malaria parasites.

Low efficiency upconversion nanoparticles for high-resolution coalignment of near-infrared and visible light paths on a light microscope

PubMed Central

Sundaramoorthy, Sriramkumar; Badaracco, Adrian Garcia; Hirsch, Sophia M.; Park, Jun Hong; Davies, Tim; Dumont, Julien; Shirasu-Hiza, Mimi; Kummel, Andrew C.; Canman, Julie C.

2017-01-01

The combination of near infrared (NIR) and visible wavelengths in light microscopy for biological studies is increasingly common. For example, many fields of biology are developing the use of NIR for optogenetics, in which an NIR laser induces a change in gene expression and/or protein function. One major technical barrier in working with both NIR and visible light on an optical microscope is obtaining their precise coalignment at the imaging plane position. Photon upconverting particles (UCPs) can bridge this gap as they are excited by NIR light but emit in the visible range via an anti-Stokes luminescence mechanism. Here, two different UCPs have been identified, high-efficiency micro540-UCPs and lower efficiency nano545-UCPs, that respond to NIR light and emit visible light with high photostability even at very high NIR power densities (>25,000 Suns). Both of these UCPs can be rapidly and reversibly excited by visible and NIR light and emit light at visible wavelengths detectable with standard emission settings used for Green Fluorescent Protein (GFP), a commonly used genetically-encoded fluorophore. However, the high efficiency micro540-UCPs were suboptimal for NIR and visible light coalignment, due to their larger size and spatial broadening from particle-to-particle energy transfer consistent with a long lived excited state and saturated power dependence. In contrast, the lower efficiency nano-UCPs were superior for precise coalignment of the NIR beam with the visible light path (~2 µm versus ~8 µm beam broadening respectively) consistent with limited particle-to-particle energy transfer, superlinear power dependence for emission, and much smaller particle size. Furthermore, the nano-UCPs were superior to a traditional two-camera method for NIR and visible light path alignment in an in vivo Infrared-Laser-Evoked Gene Operator (IR-LEGO) optogenetics assay in the budding yeast S. cerevisiae. In summary, nano-UCPs are powerful new tools for coaligning NIR and visible light paths on a light microscope. PMID:28221018

Microscopic Analysis of Activated Sludge. Training Manual.

ERIC Educational Resources Information Center

Office of Water Program Operations (EPA), Cincinnati, OH. National Training and Operational Technology Center.

This training manual presents material on the use of a compound microscope to analyze microscope communities, present in wastewater treatment processes, for operational control. Course topics include: sampling techniques, sample handling, laboratory analysis, identification of organisms, data interpretation, and use of the compound microscope.…

HIGH TEMPERATURE MICROSCOPE AND FURNACE

DOEpatents

Olson, D.M.

1961-01-31

A high-temperature microscope is offered. It has a reflecting optic situated above a molten specimen in a furnace and reflecting the image of the same downward through an inert optic member in the floor of the furnace, a plurality of spaced reflecting plane mirrors defining a reflecting path around the furnace, a standard microscope supported in the path of and forming the end terminus of the light path.

Colonization of cashew plants by Lasiodiplodia theobromae: Microscopical features

USDA-ARS?s Scientific Manuscript database

Lasiodiplodia theobromae is a phytopathogenic fungus causing gummosis, a threatening disease for cashew plants in Brazil. In an attempt to investigate the ultrastructural features of the pathogen colonization and its response to immunofluorescence labeling, light, confocal and electron microscope st...

Fluorescence-guided resection of intracranial VX2 tumor in a preclinical model using 5-aminolevulinic acid (ALA): preliminary results

NASA Astrophysics Data System (ADS)

Bogaards, Arjen; Varma, Abhay; Moriyama, Eduardo H.; Lin, Annie; Giles, Anoja; Bisland, Stuart K.; Lilge, Lothar D.; Bilbao, G. M.; Muller, Paul J.; Wilson, Brian C.

2003-06-01

Fluorescence-guided brain tumor resection may help the neurosurgeon to identify tumor margins that merge imperceptibly into the normal brain tissue and are difficult to identify under white light illumination even using an operating microscope. We compared the amount of residual tumor after white light resection using an operating microscope versus that after fluorescnece-guided resection of an intracranial VX2 tumor in a preclinical model using our previously developed co-axial fluorscence imaging and spectroscopy system, exciting and detecting PpIX fluorescence at 405nm and 635nm respectively. Preliminary results: No fluorescence was present in 3 non-tumor-bearing animals. Fluorescence was present in all 15 tumor-bearing animals after white light resection was completed. To date in 4 rabbits, a decrease in residual tumor was found when using additional fluorescence guided resection compared to white light resection only. Conclusions: ALA induced PpIX fluorescence detects tumor margins not seen under an operation microscope using while light. Using fluorescence imaging to guide tumor resection resulted in a 3-fold decrease in the amount of residual timor. However, these preliminary results indicate that also an additional amount of normal brain is resected, which will be further investigated.

High-Bandwidth Dynamic Full-Field Profilometry for Nano-Scale Characterization of MEMS

NASA Astrophysics Data System (ADS)

Chen, Liang-Chia; Huang, Yao-Ting; Chang, Pi-Bai

2006-10-01

The article describes an innovative optical interferometric methodology to delivery dynamic surface profilometry with a measurement bandwidth up to 10MHz or higher and a vertical resolution up to 1 nm. Previous work using stroboscopic microscopic interferometry for dynamic characterization of micro (opto)electromechanical systems (M(O)EMS) has been limited in measurement bandwidth mainly within a couple of MHz. For high resonant mode analysis, the stroboscopic light pulse is insufficiently short to capture the moving fringes from dynamic motion of the detected structure. In view of this need, a microscopic prototype based on white-light stroboscopic interferometry with an innovative light superposition strategy was developed to achieve dynamic full-field profilometry with a high measurement bandwidth up to 10MHz or higher. The system primarily consists of an optical microscope, on which a Mirau interferometric objective embedded with a piezoelectric vertical translator, a high-power LED light module with dual operation modes and light synchronizing electronics unit are integrated. A micro cantilever beam used in AFM was measured to verify the system capability in accurate characterisation of dynamic behaviours of the device. The full-field seventh-mode vibration at a vibratory frequency of 3.7MHz can be fully characterized and nano-scale vertical measurement resolution as well as tens micrometers of vertical measurement range can be performed.

Development of a Hybrid Atomic Force Microscopic Measurement System Combined with White Light Scanning Interferometry

PubMed Central

Guo, Tong; Wang, Siming; Dorantes-Gonzalez, Dante J.; Chen, Jinping; Fu, Xing; Hu, Xiaotang

2012-01-01

A hybrid atomic force microscopic (AFM) measurement system combined with white light scanning interferometry for micro/nanometer dimensional measurement is developed. The system is based on a high precision large-range positioning platform with nanometer accuracy on which a white light scanning interferometric module and an AFM head are built. A compact AFM head is developed using a self-sensing tuning fork probe. The head need no external optical sensors to detect the deflection of the cantilever, which saves room on the head, and it can be directly fixed under an optical microscopic interferometric system. To enhance the system’s dynamic response, the frequency modulation (FM) mode is adopted for the AFM head. The measuring data can be traceable through three laser interferometers in the system. The lateral scanning range can reach 25 mm × 25 mm by using a large-range positioning platform. A hybrid method combining AFM and white light scanning interferometry is proposed to improve the AFM measurement efficiency. In this method, the sample is measured firstly by white light scanning interferometry to get an overall coarse morphology, and then, further measured with higher resolution by AFM. Several measuring experiments on standard samples demonstrate the system’s good measurement performance and feasibility of the hybrid measurement method. PMID:22368463

Development of a hybrid atomic force microscopic measurement system combined with white light scanning interferometry.

PubMed

Guo, Tong; Wang, Siming; Dorantes-Gonzalez, Dante J; Chen, Jinping; Fu, Xing; Hu, Xiaotang

2012-01-01

A hybrid atomic force microscopic (AFM) measurement system combined with white light scanning interferometry for micro/nanometer dimensional measurement is developed. The system is based on a high precision large-range positioning platform with nanometer accuracy on which a white light scanning interferometric module and an AFM head are built. A compact AFM head is developed using a self-sensing tuning fork probe. The head need no external optical sensors to detect the deflection of the cantilever, which saves room on the head, and it can be directly fixed under an optical microscopic interferometric system. To enhance the system's dynamic response, the frequency modulation (FM) mode is adopted for the AFM head. The measuring data can be traceable through three laser interferometers in the system. The lateral scanning range can reach 25 mm × 25 mm by using a large-range positioning platform. A hybrid method combining AFM and white light scanning interferometry is proposed to improve the AFM measurement efficiency. In this method, the sample is measured firstly by white light scanning interferometry to get an overall coarse morphology, and then, further measured with higher resolution by AFM. Several measuring experiments on standard samples demonstrate the system's good measurement performance and feasibility of the hybrid measurement method.

The e-evolution of microscopy in dental education.

PubMed

Farah, Camile S; Maybury, Terrence S

2009-08-01

Recent technological innovation has now made it possible to turn the computer into a microscope. This has entailed a shift from light microscopy to virtual microscopy. This development then foregrounds the issue of the pedagogy involved in this move from the analogue technology of the light microscope to the digital, computerized instance of virtual microscopy. In order to address this issue, undergraduate students enrolled in the Bachelor of Dental Science program at the University of Queensland School of Dentistry were surveyed to ascertain their preference for light or virtual microscopy. The value of this study is that it was conducted on the same cohort of students in two separate courses in 2006 and 2008, giving it longitudinal validity. The responses were overwhelmingly in favor of virtual microscopy. When it came to completely replacing the light microscope with virtual microscopy, however, students were much more ambivalent about such a wholesale change although this was less of an issue in the senior year. This shift from light to virtual microscopy signals larger changes in the tertiary sector from print-literate to electronic forms of knowledge and from teacher-centered to student-focused frames of learning. In short, we are in the midst of the e-evolution of microscopy in dental education.

Extended morphological processing: a practical method for automatic spot detection of biological markers from microscopic images.

PubMed

Kimori, Yoshitaka; Baba, Norio; Morone, Nobuhiro

2010-07-08

A reliable extraction technique for resolving multiple spots in light or electron microscopic images is essential in investigations of the spatial distribution and dynamics of specific proteins inside cells and tissues. Currently, automatic spot extraction and characterization in complex microscopic images poses many challenges to conventional image processing methods. A new method to extract closely located, small target spots from biological images is proposed. This method starts with a simple but practical operation based on the extended morphological top-hat transformation to subtract an uneven background. The core of our novel approach is the following: first, the original image is rotated in an arbitrary direction and each rotated image is opened with a single straight line-segment structuring element. Second, the opened images are unified and then subtracted from the original image. To evaluate these procedures, model images of simulated spots with closely located targets were created and the efficacy of our method was compared to that of conventional morphological filtering methods. The results showed the better performance of our method. The spots of real microscope images can be quantified to confirm that the method is applicable in a given practice. Our method achieved effective spot extraction under various image conditions, including aggregated target spots, poor signal-to-noise ratio, and large variations in the background intensity. Furthermore, it has no restrictions with respect to the shape of the extracted spots. The features of our method allow its broad application in biological and biomedical image information analysis.

Single particle tracking through highly scattering media with multiplexed two-photon excitation

NASA Astrophysics Data System (ADS)

Perillo, Evan; Liu, Yen-Liang; Liu, Cong; Yeh, Hsin-Chih; Dunn, Andrew K.

2015-03-01

3D single-particle tracking (SPT) has been a pivotal tool to furthering our understanding of dynamic cellular processes in complex biological systems, with a molecular localization accuracy (10-100 nm) often better than the diffraction limit of light. However, current SPT techniques utilize either CCDs or a confocal detection scheme which not only suffer from poor temporal resolution but also limit tracking to a depth less than one scattering mean free path in the sample (typically <15μm). In this report we highlight our novel design for a spatiotemporally multiplexed two-photon microscope which is able to reach sub-diffraction-limit tracking accuracy and sub-millisecond temporal resolution, but with a dramatically extended SPT range of up to 200 μm through dense cell samples. We have validated our microscope by tracking (1) fluorescent nanoparticles in a prescribed motion inside gelatin gel (with 1% intralipid) and (2) labeled single EGFR complexes inside skin cancer spheroids (at least 8 layers of cells thick) for ~10 minutes. Furthermore we discuss future capabilities of our multiplexed two-photon microscope design, specifically to the extension of (1) simultaneous multicolor tracking (i.e. spatiotemporal co-localization analysis) and (2) FRET studies (i.e. lifetime analysis). The high resolution, high depth penetration, and multicolor features of this microscope make it well poised to study a variety of molecular scale dynamics in the cell, especially related to cellular trafficking studies with in vitro tumor models and in vivo.

Microscopic heat pulses induce contraction of cardiomyocytes without calcium transients

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

Oyama, Kotaro; Mizuno, Akari; Shintani, Seine A.

Highlights: Black-Right-Pointing-Pointer Infra-red laser beam generates microscopic heat pulses. Black-Right-Pointing-Pointer Heat pulses induce contraction of cardiomyocytes. Black-Right-Pointing-Pointer Ca{sup 2+} transients during the contraction were not detected. Black-Right-Pointing-Pointer Skinned cardiomyocytes in free Ca{sup 2+} solution also contracted. Black-Right-Pointing-Pointer Heat pulses regulated the contractions without Ca{sup 2+} dynamics. -- Abstract: It was recently demonstrated that laser irradiation can control the beating of cardiomyocytes and hearts, however, the precise mechanism remains to be clarified. Among the effects induced by laser irradiation on biological tissues, temperature change is one possible effect which can alter physiological functions. Therefore, we investigated the mechanism by which heatmore » pulses, produced by infra-red laser light under an optical microscope, induce contractions of cardiomyocytes. Here we show that microscopic heat pulses induce contraction of rat adult cardiomyocytes. The temperature increase, {Delta}T, required for inducing contraction of cardiomyocytes was dependent upon the ambient temperature; that is, {Delta}T at physiological temperature was lower than that at room temperature. Ca{sup 2+} transients, which are usually coupled to contraction, were not detected. We confirmed that the contractions of skinned cardiomyocytes were induced by the heat pulses even in free Ca{sup 2+} solution. This heat pulse-induced Ca{sup 2+}-decoupled contraction technique has the potential to stimulate heart and skeletal muscles in a manner different from the conventional electrical stimulations.« less

A simple apparatus for quick qualitative analysis of CR39 nuclear track detectors

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

Gautier, D. C.; Kline, J. L.; Flippo, K. A.

2008-10-15

Quantifying the ion pits in Columbia Resin 39 (CR39) nuclear track detector from Thomson parabolas is a time consuming and tedious process using conventional microscope based techniques. A simple inventive apparatus for fast screening and qualitative analysis of CR39 detectors has been developed, enabling efficient selection of data for a more detailed analysis. The system consists simply of a green He-Ne laser and a high-resolution digital single-lens reflex camera. The laser illuminates the edge of the CR39 at grazing incidence and couples into the plastic, acting as a light pipe. Subsequently, the laser illuminates all ion tracks on the surface.more » A high-resolution digital camera is used to photograph the scattered light from the ion tracks, enabling one to quickly determine charge states and energies measured by the Thomson parabola.« less

Visual Identification of Light-Driven Breakage of the Silver-Dithiocarbamate Bond by Single Plasmonic Nanoprobes

PubMed Central

Gao, Peng Fei; Yuan, Bin Fang; Gao, Ming Xuan; Li, Rong Sheng; Ma, Jun; Zou, Hong Yan; Li, Yuan Fang; Li, Ming; Huang, Cheng Zhi

2015-01-01

Insight into the nature of metal-sulfur bond, a meaningful one in life science, interface chemistry and organometallic chemistry, is interesting but challenging. By utilizing the localized surface plasmon resonance properties of silver nanoparticles, herein we visually identified the photosensitivity of silver-dithiocarbamate (Ag-DTC) bond by using dark field microscopic imaging (iDFM) technique at single nanoparticle level. It was found that the breakage of Ag-DTC bond could be accelerated effectively by light irradiation, followed by a pH-dependent horizontal or vertical degradation of the DTC molecules, in which an indispensable preoxidation process of the silver was at first disclosed. These findings suggest a visualization strategy at single plasmonic nanoparticle level which can be excellently applied to explore new stimulus-triggered reactions, and might also open a new way to understand traditional organic reaction mechanisms. PMID:26493773

Real-Time Light Scattering Tracking of Gold Nanoparticles- bioconjugated Respiratory Syncytial Virus Infecting HEp-2 Cells

NASA Astrophysics Data System (ADS)

Wan, Xiao-Yan; Zheng, Lin-Ling; Gao, Peng-Fei; Yang, Xiao-Xi; Li, Chun-Mei; Li, Yuan Fang; Huang, Cheng Zhi

2014-03-01

Real-time tracking of virus invasion is crucial for understanding viral infection mechanism, which, however, needs simple and efficient labeling chemistry with improved signal-to-noise ratio. For that purpose, herein we investigated the invasion dynamics of respiratory syncytial virus (RSV) through dark-field microscopic imaging (iDFM) technique by using Au nanoparticles (AuNPs) as light scattering labels. RSV, a ubiquitous, non-segmented, pleiomorphic and negative-sense RNA virus, is an important human pathogen in infants, the elderly, and the immunocompromised. In order to label the enveloped virus of paramyxoviridae family, an efficient streptavidin (SA)-biotin binding chemistry was employed, wherein AuNPs and RSV particles modified with SA and biotin, respectively, allowing the AuNP-modified RSVs to maintain their virulence without affecting the native activities of RSV, making the long dynamic visualization successful for the RSV infections into human epidermis larynx carcinoma cells.

Photo-induced ultrasound microscopy for photo-acoustic imaging of non-absorbing specimens

NASA Astrophysics Data System (ADS)

Tcarenkova, Elena; Koho, Sami V.; Hänninen, Pekka E.

2017-08-01

Photo-Acoustic Microscopy (PAM) has raised high interest in in-vivo imaging due to its ability to preserve the near-diffraction limited spatial resolution of optical microscopes, whilst extending the penetration depth to the mm-range. Another advantage of PAM is that it is a label-free technique - any substance that absorbs PAM excitation laser light can be viewed. However, not all sample structures desired to be observed absorb sufficiently to provide contrast for imaging. This work describes a novel imaging method that makes it possible to visualize optically transparent samples that lack intrinsic photo-acoustic contrast, without the addition of contrast agents. A thin, strongly light absorbing layer next to sample is used to generate a strong ultrasonic signal. This signal, when recorded from opposite side, contains ultrasonic transmission information of the sample and thus the method can be used to obtain an ultrasound transmission image on any PAM.

Digital photocontrol of the network of live excitable cells

NASA Astrophysics Data System (ADS)

Erofeev, I. S.; Magome, N.; Agladze, K. I.

2011-11-01

Recent development of tissue engineering techniques allows creating and maintaining almost indefinitely networks of excitable cells with desired architecture. We coupled the network of live excitable cardiac cells with a common computer by sensitizing them to light, projecting a light pattern on the layer of cells, and monitoring excitation with the aid of fluorescent probes (optical mapping). As a sensitizing substance we used azobenzene trimethylammonium bromide (AzoTAB). This substance undergoes cis-trans-photoisomerization and trans-isomer of AzoTAB inhibits excitation in the cardiac cells, while cis-isomer does not. AzoTAB-mediated sensitization allows, thus, reversible and dynamic control of the excitation waves through the entire cardiomyocyte network either uniformly, or in a preferred spatial pattern. Technically, it was achieved by coupling a common digital projector with a macroview microscope and using computer graphic software for creating the projected pattern of conducting pathways. This approach allows real time interactive photocontrol of the heart tissue.

Biological soft X-ray tomography on beamline 2.1 at the Advanced Light Source.

PubMed

Le Gros, Mark A; McDermott, Gerry; Cinquin, Bertrand P; Smith, Elizabeth A; Do, Myan; Chao, Weilun L; Naulleau, Patrick P; Larabell, Carolyn A

2014-11-01

Beamline 2.1 (XM-2) is a transmission soft X-ray microscope in sector 2 of the Advanced Light Source at Lawrence Berkeley National Laboratory. XM-2 was designed, built and is now operated by the National Center for X-ray Tomography as a National Institutes of Health Biomedical Technology Research Resource. XM-2 is equipped with a cryogenic rotation stage to enable tomographic data collection from cryo-preserved cells, including large mammalian cells. During data collection the specimen is illuminated with `water window' X-rays (284-543 eV). Illuminating photons are attenuated an order of magnitude more strongly by biomolecules than by water. Consequently, differences in molecular composition generate quantitative contrast in images of the specimen. Soft X-ray tomography is an information-rich three-dimensional imaging method that can be applied either as a standalone technique or as a component modality in correlative imaging studies.

PDMS microfludic device for optical detection of protein immunoassay using gold nanoparticles.

PubMed

Luo, Chunxiong; Fu, Qiang; Li, Hao; Xu, Luping; Sun, Manhui; Ouyang, Qi; Chen, Yong; Ji, Hang

2005-07-01

A simple but highly specific immunoassay system for goat anti-human IgG has been developed using gold nanoparticles and microfluidic techniques. The assay is based on the deposition of gold nanoparticles that are coated with protein antigens in the presence of their corresponding antibodies to microfluidic channel surface. The effects of time accumulation, the flow velocity, and the concentration of antibodies to the red light absorption percentage (RAP) of deposition were investigated with an ordinary optical microscope. By controlling the reaction time and flow velocity, a dynamic range of 3 orders of magnitude and a detection sensitivity of 10 ng ml(-1) of goat anti-human IgG were achieved. Because of its simplicity and flexibility, this new technique should be useful for fast, highthroughput screening of antibodies in clinical diagnostic applications.

Facile synthesis of SiO{sub 2} nanoparticles for biomedical applications

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

Scano, A., E-mail: alescano80@tiscali.it; Pilloni, M., E-mail: alescano80@tiscali.it; Cabras, V., E-mail: alescano80@tiscali.it

Silica nanoparticles (SiO{sub 2} NPs) for biomedical applications have been prepared by using a facile modified Stöber-synthesis. Potassium borohydride (KBH{sub 4}) has been introduced in the synthesis procedure in order to control NP size. Several samples have been prepared varying tetraethylorthosilicate (TEOS) concentration, and using different process conditions (temperature, reaction time and atmosphere). In order to study the influence of the process conditions on the NP size, morphology and properties, several characterization techniques were used. Size and morphology of the as-prepared SiO{sub 2} NPs have been studied by using Transmission Electron Microscope (TEM) and Dynamic Light Scattering (DLS) techniques. Structuralmore » characterization was carried out by X-ray powder diffraction. To investigate the SiO{sub 2} NP fluorescence emission properties the fluorescence spectroscopy was also used.« less

Effect of etching time on morphological, optical, and electronic properties of silicon nanowires

PubMed Central

2012-01-01

Owing to their interesting electronic, mechanical, optical, and transport properties, silicon nanowires (SiNWs) have attracted much attention, giving opportunities to several potential applications in nanoscale electronic, optoelectronic devices, and silicon solar cells. For photovoltaic application, a superficial film of SiNWs could be used as an efficient antireflection coating. In this work we investigate the morphological, optical, and electronic properties of SiNWs fabricated at different etching times. Characterizations of the formed SiNWs films were performed using a scanning electron microscope, ultraviolet–visible-near-infrared spectroscopy, and light-beam-induced-current technique. The latter technique was used to determine the effective diffusion length in SiNWs films. From these investigations, we deduce that the homogeneity of the SiNWs film plays a key role on the electronic properties. PMID:22799265

Role of coherence in microsphere-assisted nanoscopy

NASA Astrophysics Data System (ADS)

Perrin, Stephane; Lecler, Sylvain; Leong-Hoi, Audrey; Montgomery, Paul C.

2017-06-01

The loss of the information, due to the diffraction and the evanescent waves, limits the resolving power of classical optical microscopy. In air, the lateral resolution of an optical microscope can approximated at half of the wavelength using a low-coherence illumination. Recently, several methods have been developed in order to overcome this limitation and, in 2011, a new far-field and full-field imaging technique was proposed where a sub-diffraction-limit resolution has been achieved using a transparent microsphere. In this article, the phenomenon of super-resolution using microsphere-assisted microscopy is analysed through rigorous electro-magnetic simulations. The performances of the imaging technique are estimated as function of optical and geometrical parameters. Furthermore, the role of coherence is introduced through the temporal coherence of the light source and the phase response of the object.

Energy and Technology Review

NASA Astrophysics Data System (ADS)

Poggio, Andrew J.

1988-10-01

This issue of Energy and Technology Review contains: Neutron Penumbral Imaging of Laser-Fusion Targets--using our new penumbral-imaging diagnostic, we have obtained the first images that can be used to measure directly the deuterium-tritium burn region in laser-driven fusion targets; Computed Tomography for Nondestructive Evaluation--various computed tomography systems and computational techniques are used in nondestructive evaluation; Three-Dimensional Image Analysis for Studying Nuclear Chromatin Structure--we have developed an optic-electronic system for acquiring cross-sectional views of cell nuclei, and computer codes to analyze these images and reconstruct the three-dimensional structures they represent; Imaging in the Nuclear Test Program--advanced techniques produce images of unprecedented detail and resolution from Nevada Test Site data; and Computational X-Ray Holography--visible-light experiments and numerically simulated holograms test our ideas about an X-ray microscope for biological research.

Direction-division multiplexed holographic free-electron-driven light sources

NASA Astrophysics Data System (ADS)

Clarke, Brendan P.; MacDonald, Kevin F.; Zheludev, Nikolay I.

2018-01-01

We report on a free-electron-driven light source with a controllable direction of emission. The source comprises a microscopic array of plasmonic surface-relief holographic domains, each tailored to direct electron-induced light emission at a selected wavelength into a collimated beam in a prescribed direction. The direction-division multiplexed source is tested by driving it with the 30 kV electron beam of a scanning electron microscope: light emission, at a wavelength of 800 nm in the present case, is switched among different output angles by micron-scale repositioning of the electron injection point among domains. Such sources, with directional switching/tuning possible at picosecond timescales, may be applied to field-emission and surface-conduction electron-emission display technologies, optical multiplexing, and charged-particle-beam position metrology.

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

Gupta, S.; Arend, N.; Lunkenheimer, P.

The relaxational dynamics in glass-forming glycerol and glycerol mixed with LiCl is investigated using different neutron scattering techniques. The performed neutron spin echo experiments, which extend up to relatively long relaxation time scales of the order of 10 ns, should allow for the detection of contributions from the so-called excess wing. This phenomenon, whose microscopic origin is controversially discussed, arises in a variety of glass formers and, until now, was almost exclusively investigated by dielectric spectroscopy and light scattering. In conclusion, we show here that the relaxational process causing the excess wing can also be detected by neutron scattering, whichmore » directly couples to density fluctuations.« less

Fluorescein sodium-guided surgery of a brain abscess: A case report

PubMed Central

Höhne, Julius; Brawanski, Alexander; Schebesch, Karl-Michael

2016-01-01

Background: Up to now, the feasibility and benefit of using fluorescein sodium under a dedicated surgical microscope filter (YE560, YELLOW 560 nm filter, Carl Zeiss Meditec, Germany) has never been clinically evaluated in infectious disorders of the brain. Case Description: Here, we report the case of a male patient with a brain abscess in the right parietal lobe that was removed under fluorescence-guidance (intravenous administration of fluorescein sodium 10%, 5 mg/kg bodyweight). The abscess capsule showed intensive yellow fluorescent staining, while − under white light − the cortex appeared normal. Conclusion: This technique may improve the identification and surgical removal of brain abscesses. PMID:28031990

Polarized light and scanning electron microscopic investigation of enamel hypoplasia in primary teeth.

PubMed

Sabel, Nina; Klingberg, Gunilla; Dietz, Wolfram; Nietzsche, Sandor; Norén, Jörgen G

2010-01-01

Enamel hypoplasia is a developmental disturbance during enamel formation, defined as a macroscopic defect in the enamel, with a reduction of the enamel thickness with rounded, smooth borders. Information on the microstructural level is still limited, therefore further studies are of importance to better understand the mechanisms behind enamel hypoplasia. To study enamel hypoplasia in primary teeth by means of polarized light microscopy and scanning electron microscopy. Nineteen primary teeth with enamel hypoplasia were examined in a polarized light microscope and in a scanning electron microscope. The cervical and incisal borders of the enamel hypoplasia had a rounded appearance, as the prisms in the rounded cervical area of the hypoplasia were bent. The rounded borders had a normal surface structure whereas the base of the defects appeared rough and porous. Morphological findings in this study indicate that the aetiological factor has a short duration and affects only certain ameloblasts. The bottom of the enamel hypoplasia is porous and constitutes possible pathways for bacteria into the dentin.

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

Timmermans, F. J.; Otto, C.

New developments in the field of microscopy enable to acquire increasing amounts of information from large sample areas and at an increased resolution. Depending on the nature of the technique, the information may reveal morphological, structural, chemical, and still other sample characteristics. In research fields, such as cell biology and materials science, there is an increasing demand to correlate these individual levels of information and in this way to obtain a better understanding of sample preparation and specific sample properties. To address this need, integrated systems were developed that combine nanometer resolution electron microscopes with optical microscopes, which produce chemicallymore » or label specific information through spectroscopy. The complementary information from electron microscopy and light microscopy presents an opportunity to investigate a broad range of sample properties in a correlated fashion. An important part of correlating the differences in information lies in bridging the different resolution and image contrast features. The trend to analyse samples using multiple correlated microscopes has resulted in a new research field. Current research is focused, for instance, on (a) the investigation of samples with nanometer scale distribution of inorganic and organic materials, (b) live cell analysis combined with electron microscopy, and (c) in situ spectroscopic and electron microscopy analysis of catalytic materials, but more areas will benefit from integrated correlative microscopy.« less

Measurement of glomerulus diameter and Bowman's space width of renal albino rats.

PubMed

Kotyk, Taras; Dey, Nilanjan; Ashour, Amira S; Balas-Timar, Dana; Chakraborty, Sayan; Ashour, Ahmed S; Tavares, João Manuel R S

2016-04-01

Glomerulus diameter and Bowman's space width in renal microscopic images indicate various diseases. Therefore, the detection of the renal corpuscle and related objects is a key step in histopathological evaluation of renal microscopic images. However, the task of automatic glomeruli detection is challenging due to their wide intensity variation, besides the inconsistency in terms of shape and size of the glomeruli in the renal corpuscle. Here, a novel solution is proposed which includes the Particles Analyzer technique based on median filter for morphological image processing to detect the renal corpuscle objects. Afterwards, the glomerulus diameter and Bowman's space width are measured. The solution was tested with a dataset of 21 rats' renal corpuscle images acquired using light microscope. The experimental results proved that the proposed solution can detect the renal corpuscle and its objects efficiently. As well as, the proposed solution has the ability to manage any input images assuring its robustness to the deformations of the glomeruli even with the glomerular hypertrophy cases. Also, the results reported significant difference between the control and affected (due to ingested additional daily dose (14.6mg) of fructose) groups in terms of glomerulus diameter (97.40±19.02μm and 177.03±54.48μm, respectively). Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Label-free imaging of intracellular motility by low-coherent quantitative phase microscope in reflection geometry

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Iwai, Hidenao; Yamashita, Yutaka

2011-11-01

We demonstrate tomographic imaging of intracellular activity of living cells by a low-coherent quantitative phase microscope. The intracellular organelles, such as the nucleus, nucleolus, and mitochondria, are moving around inside living cells, driven by the cellular physiological activity. In order to visualize the intracellular motility in a label-free manner we have developed a reflection-type quantitative phase microscope which employs the phase shifting interferometric technique with a low-coherent light source. The phase shifting interferometry enables us to quantitatively measure the intensity and phase of the optical field, and the low-coherence interferometry makes it possible to selectively probe a specific sectioning plane in the cell volume. The results quantitatively revealed the depth-resolved fluctuations of intracellular surfaces so that the plasma membrane and the membranes of intracellular organelles were independently measured. The transversal and the vertical spatial resolutions were 0.56 μm and 0.93 μm, respectively, and the mechanical sensitivity of the phase measurement was 1.2 nanometers. The mean-squared displacement was applied as a statistical tool to analyze the temporal fluctuation of the intracellular organelles. To the best of our knowledge, our system visualized depth-resolved intracellular organelles motion for the first time in sub-micrometer resolution without contrast agents.

A STED-FLIM microscope applied to imaging the natural killer cell immune synapse

NASA Astrophysics Data System (ADS)

Lenz, M. O.; Brown, A. C. N.; Auksorius, E.; Davis, D. M.; Dunsby, C.; Neil, M. A. A.; French, P. M. W.

2011-03-01

We present a stimulated emission depletion (STED) fluorescence lifetime imaging (FLIM) microscope, excited by a microstructured optical fibre supercontinuum source that is pumped by a femtosecond Ti:Sapphire-laser, which is also used for depletion. Implemented using a piezo-scanning stage on a laser scanning confocal fluorescence microscope system with FLIM realised using time correlated single photon counting (TCSPC), this provides convenient switching between confocal and STED-FLIM with spatial resolution down to below 60 nm. We will present our design considerations to make a robust instrument for biological applications including a comparison between fixed phase plate and spatial light modulator (SLM) approaches to shape the STED beam and the correlation of STED and confocal FLIM microscopy. Following our previous application of FLIM-FRET to study intercellular signalling at the immunological synapse (IS), we are employing STED microscopy to characterize the spatial distribution of cellular molecules with subdiffraction resolution at the IS. In particular, we are imaging cytoskeletal structure at the Natural Killer cell activated immune synapse. We will also present our progress towards multilabel STED microscopy to determine how relative spatial molecular organization, previously undetectable by conventional microscopy techniques, is important for NK cell cytotoxic function. Keywords: STED, Stimulated Emission Depletion Microscopy, Natural Killer (NK) cell, Fluorescence lifetime imaging, FLIM, Super resolution microscopy.

Determining the phonon energy of highly oriented pyrolytic graphite by scanning tunneling microscope light emission spectroscopy

NASA Astrophysics Data System (ADS)

Uehara, Yoichi; Michimata, Junichi; Watanabe, Shota; Katano, Satoshi; Inaoka, Takeshi

2018-03-01

We have investigated the scanning tunneling microscope (STM) light emission spectra of isolated single Ag nanoparticles lying on highly oriented pyrolytic graphite (HOPG). The STM light emission spectra exhibited two types of spectral structures (step-like and periodic). Comparisons of the observed structures and theoretical predictions indicate that the phonon energy of the ZO mode of HOPG [M. Mohr et al., Phys. Rev. B 76, 035439 (2007)] can be determined from the energy difference between the cutoff of STM light emission and the step in the former structure, and from the period of the latter structure. Since the role of the Ag nanoparticles does not depend on the substrate materials, this method will enable the phonon energies of various materials to be measured by STM light emission spectroscopy. The spatial resolution is comparable to the lateral size of the individual Ag nanoparticles (that is, a few nm).

Plasmon-resonance-enhanced visible-light photocatalytic activity of Ag quantum dots/TiO2 microspheres for methyl orange degradation

NASA Astrophysics Data System (ADS)

Yu, Xin; Shang, Liwei; Wang, Dongjun; An, Li; Li, Zhonghua; Liu, Jiawen; Shen, Jun

2018-06-01

We successfully prepared Ag quantum dots modified TiO2 microspheres by facile solvothermal and calcination method. The as-prepared Ag quantum dots/TiO2 microspheres were characterized by scanning electron microscope, transmission electron microscope, X-ray diffraction, X-ray photoelectron spectroscopy and UV-vis diffuse reflectance spectroscopy. The Ag quantum dots/TiO2 photocatalyst showed excellent visible light absorption and efficient photocatalytic activity for methyl orange degradation. And the sample with the molar ratio of 0.05 (Ag to Ti) showed the best visible light photocatalytic activity for methyl orange degradation, mainly because of the surface plasmon resonance (SPR) effects of Ag quantum dots to generate electron and hole pairs for enhanced visible light photocatalysis. Finally, possible visible light photocatalytic mechanism of Ag quantum dots/TiO2 microspheres for methyl orange degradation was proposed in detail.

A Mach-Zender Holographic Microscope for Quantifying Bacterial Motility

NASA Astrophysics Data System (ADS)

Niraula, B.; Nadeau, J. L.; Serabyn, E.; Wallace, J. K.; Liewer, K.; Kuhn, J.; Graff, E.; Lindensmith, C.

2014-12-01

New microscopic techniques have revolutionized cell biology over the past two decades. However, there are still biological processes whose details elude us, especially those involving motility: e.g. feeding behavior of microorganisms in the ocean, or migration of cancer cells to form metastases. Imaging prokaryotes, which range in size from several hundred nm to a few microns, is especially challenging. An emerging technique to address these issues is Digital Holographic Microscopy (DHM). DHM is an imaging technique that uses the interference of light to record and reproduce three-dimensional magnified images of objects. This approach has several advantages over ordinary brightfield microscopy for fieldwork: a larger depth of field, hands-off operation, robustness regarding environmental conditions, and large sampling volumes with quantitative 3D records of motility behavior. Despite these promising features, real-time DHM was thought to be impractical for technological and computational reasons until recently, and there has so far been very limited application of DHM to biology. Most existing instruments are limited in performance by their particular (e.g. in-line, lens-less, phase-shifting) approach to holography. These limitations can be mitigated with an off-axis dual-path configuration. Here we describe the design and implementation of a design for a Mach-Zehnder-type holographic microscope with diffraction-limited lateral resolution, with intended applications in environmental microbiology. We have achieved sub-micron resolution and three-dimensional tracking of prokaryotic and eukaryotic test strains designed to represent different modes and speeds of microbial motility. Prokaryotes are Escherichia coli, Vibrio alginolyticus, and Bacillus subtilis. Each shows a characteristic motility pattern, as we illustrate in holographic videos in sample chambers 0.6 mm in depth. The ability to establish gradients of attractants with bacterial taxis towards the attractant is also established. The eukaryotic strains are Euglena gracilis, which demonstrates both phototaxis and geotaxis, and Paramecium micromultinucleatum. The challenges of optimizing resolution vs. field of view, and of handling the large volumes of data generated during holographic imaging, are discussed.

Comparison of Confocal and Super-Resolution Reflectance Imaging of Metal Oxide Nanoparticles

PubMed Central

Guggenheim, Emily J.; Khan, Abdullah; Pike, Jeremy; Chang, Lynne; Lynch, Iseult; Rappoport, Joshua Z.

2016-01-01

The potential for human exposure to manufactured nanoparticles (NPs) has increased in recent years, in part through the incorporation of engineered particles into a wide range of commercial goods and medical applications. NP are ideal candidates for use as therapeutic and diagnostic tools within biomedicine, however concern exists regarding their efficacy and safety. Thus, developing techniques for the investigation of NP uptake into cells is critically important. Current intracellular NP investigations rely on the use of either Transmission Electron Microscopy (TEM), which provides ultrahigh resolution, but involves cumbersome sample preparation rendering the technique incompatible with live cell imaging, or fluorescent labelling, which suffers from photobleaching, poor bioconjugation and, often, alteration of NP surface properties. Reflected light imaging provides an alternative non-destructive label free technique well suited, but not limited to, the visualisation of NP uptake within model systems, such as cells. Confocal reflectance microscopy provides optical sectioning and live imaging capabilities, with little sample preparation. However confocal microscopy is diffraction limited, thus the X-Y resolution is restricted to ~250 nm, substantially larger than the <100 nm size of NPs. Techniques such as super-resolution light microscopy overcome this fundamental limitation, providing increased X-Y resolution. The use of Reflectance SIM (R-SIM) for NP imaging has previously only been demonstrated on custom built microscopes, restricting the widespread use and limiting NP investigations. This paper demonstrates the use of a commercial SIM microscope for the acquisition of super-resolution reflectance data with X-Y resolution of 115 nm, a greater than two-fold increase compared to that attainable with RCM. This increase in resolution is advantageous for visualising small closely spaced structures, such as NP clusters, previously unresolvable by RCM. This is advantageous when investigating the subcellular trafficking of NP within fluorescently labelled cellular compartments. NP signal can be observed using RCM, R-SIM and TEM and a direct comparison is presented. Each of these techniques has its own benefits and limitations; RCM and R-SIM provide novel complementary information while the combination of modalities provides a unique opportunity to gain additional information regarding NP uptake. The use of multiple imaging methods therefore greatly enhances the range of NPs that can be studied under label-free conditions. PMID:27695038

Resonance Raman microscopy in combination with partial dark-field microscopy lights up a new path in malaria diagnostics.

PubMed

Wood, Bayden R; Hermelink, Antje; Lasch, Peter; Bambery, Keith R; Webster, Grant T; Khiavi, Mehdi Asghari; Cooke, Brian M; Deed, Samantha; Naumann, Dieter; McNaughton, Don

2009-06-01

Our goal is to produce a rapid and accurate diagnostic tool for malaria using resonance Raman spectroscopy to detect small inclusions of haemozoin in Plasmodium falciparum infected red blood cells. In pursuit of this aim we serendipitously discovered a partial dark-field effect generated by our experimental setup, which helps identify in thick blood films potential parasites that are normally difficult to see with conventional bright-field microscopy. The haemozoin deposits 'light up' and these can be selectively targeted with the Raman microscope to confirm the presence or absence of haemozoin by the strong 1569 cm(-1) band, which is a marker for haemozoin. With newly developed imaging Raman microscopes incorporating ultra-sensitive rapid readout CCDs it is possible to obtain spectra with a good signal-to-noise ratio in 1 second. Moreover, images from a smear of potentially infected cells can be recorded and analysed with multivariate methods. The reconstructed images show what appear to be sub-micron-inclusions of haemozoin in some cells indicating that the technique has potential to identify low pigmented forms of the parasite including early trophozoite-stage infected cells. Further work is required to unambiguously confirm the presence of such forms through systematic staining but the results are indeed promising and may lead to the development of a new Raman-based malaria diagnostic.

Automated measurement of diatom size

USGS Publications Warehouse

Spaulding, Sarah A.; Jewson, David H.; Bixby, Rebecca J.; Nelson, Harry; McKnight, Diane M.

2012-01-01

Size analysis of diatom populations has not been widely considered, but it is a potentially powerful tool for understanding diatom life histories, population dynamics, and phylogenetic relationships. However, measuring cell dimensions on a light microscope is a time-consuming process. An alternative technique has been developed using digital flow cytometry on a FlowCAM® (Fluid Imaging Technologies) to capture hundreds, or even thousands, of images of a chosen taxon from a single sample in a matter of minutes. Up to 30 morphological measures may be quantified through post-processing of the high resolution images. We evaluated FlowCAM size measurements, comparing them against measurements from a light microscope. We found good agreement between measurement of apical cell length in species with elongated, straight valves, including small Achnanthidium minutissimum (11-21 µm) and largeDidymosphenia geminata (87–137 µm) forms. However, a taxon with curved cells, Hannaea baicalensis (37–96 µm), showed differences of ~ 4 µm between the two methods. Discrepancies appear to be influenced by the choice of feret or geodesic measurement for asymmetric cells. We describe the operating conditions necessary for analysis of size distributions and present suggestions for optimal instrument conditions for size analysis of diatom samples using the FlowCAM. The increased speed of data acquisition through use of imaging flow cytometers like the FlowCAM is an essential step for advancing studies of diatom populations.

Evaluation of transurethral and transperineal tin ethyl etiopurpurin-photodynamic therapy on the canine prostate one week after drug injection

NASA Astrophysics Data System (ADS)

Selman, Steven H.; Keck, Rick W.; Kondo, Sandy; Albrecht, Detlef

1999-06-01

We have been investigating the potential applicability of photodynamic therapy for the treatment of benign and malignant disease of the prostate. Both transurethral and transperineal approaches to the delivery of light to the tin ethyl etiopurpurin sensitized canine prostate have been studied. Pharmacologic studies were performed and suggested that delaying light treatment for 7 days after drug administration would maximize the desired effect on the targeted prostatic tissue while minimizing the damage to surrounding bladder and rectum. A total of 12 dogs were treated with transurethral light alone (n=6) or the combination of transurethral light and transperineal light one week after tin ethyl etiopurpurin administration. (Previous studies have shown that light alone has no effect on prostate size or histology.) Animals were euthanized 48 hours and 3 weeks after completion of treatment (drug, 1mg/kg day 0, light [400mw/750sec]day 7). Tissue response was determined by gross and microscopic examination. Additionally, pre- and post- treatment transrectal ultrasounds were compared to assess changes in prostate volume and tissue echogenicity. The combination of transurethral and transperineal light results in extensive destruction of glandular epithelium with minimal damage to surrounding structures. Prostate volumes decreased by an average of 52%. Untreated areas were found to lie greater than 0.5 cm from the light diffuser. These studies have encouraged us to continue to investigate this modality as a technique for total ablation of prostatic glandular epithelium.

3D widefield light microscope image reconstruction without dyes

NASA Astrophysics Data System (ADS)

Larkin, S.; Larson, J.; Holmes, C.; Vaicik, M.; Turturro, M.; Jurkevich, A.; Sinha, S.; Ezashi, T.; Papavasiliou, G.; Brey, E.; Holmes, T.

2015-03-01

3D image reconstruction using light microscope modalities without exogenous contrast agents is proposed and investigated as an approach to produce 3D images of biological samples for live imaging applications. Multimodality and multispectral imaging, used in concert with this 3D optical sectioning approach is also proposed as a way to further produce contrast that could be specific to components in the sample. The methods avoid usage of contrast agents. Contrast agents, such as fluorescent or absorbing dyes, can be toxic to cells or alter cell behavior. Current modes of producing 3D image sets from a light microscope, such as 3D deconvolution algorithms and confocal microscopy generally require contrast agents. Zernike phase contrast (ZPC), transmitted light brightfield (TLB), darkfield microscopy and others can produce contrast without dyes. Some of these modalities have not previously benefitted from 3D image reconstruction algorithms, however. The 3D image reconstruction algorithm is based on an underlying physical model of scattering potential, expressed as the sample's 3D absorption and phase quantities. The algorithm is based upon optimizing an objective function - the I-divergence - while solving for the 3D absorption and phase quantities. Unlike typical deconvolution algorithms, each microscope modality, such as ZPC or TLB, produces two output image sets instead of one. Contrast in the displayed image and 3D renderings is further enabled by treating the multispectral/multimodal data as a feature set in a mathematical formulation that uses the principal component method of statistics.

Effect of the angle of the operating microscope light beam on visual recovery after phacoemulsification: Randomized trial.

PubMed

Harman, Francesca E; Corbett, Melanie C; Stevens, Julian D

2010-08-01

To evaluate differences in visual recovery after phacoemulsification with direct or tilted surgical microscope illumination using a macular photostress test. Western Eye Hospital, Imperial College Health Care National Health Service Trust, London, United Kingdom. This randomized double-masked controlled trial enrolled patients presenting to a daycare unit for single-eye cataract surgery. Inclusion criteria were no ocular pathology other than cataract, corneal keratometric astigmatism less than 1.50 diopters, intended target of emmetropia in the operated eye, and cataract grade 1 to 3 (Lens Opacification Classification System II). Exclusion criteria were an abnormal preoperative photostress test. Patients were randomized to have phacoemulsification with the operating microscope angled 15 degrees nasal to the fovea (study group) or with the operating microscope directly overhead around the optic disc region (control group). The same surgeon performed all phacoemulsification procedures using a standardized technique and topical anesthesia. Outcome measures were uncorrected (UDVA) and corrected (CDVA) distance visual acuity 10 minutes and 60 minutes postoperatively. In the 30 patients evaluated, the mean UDVA 10 minutes postoperatively was 0.40 logMAR +/- 0.26 (SD) in the study group and 0.72 +/- 0.36 logMAR in the control group (P<.01). The mean CDVA was 0.18 +/- 0.26 logMAR and 0.44 +/- 0.30 logMAR, respectively (P = .016). There was no significant between-group difference in acuity at 60 minutes. Tilting the microscope beam away from the fovea resulted in faster visual recovery and less macular photic stress. No author has a financial or proprietary interest in any material or method mentioned. Copyright 2010 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Microscope self-calibration based on micro laser line imaging and soft computing algorithms

NASA Astrophysics Data System (ADS)

Apolinar Muñoz Rodríguez, J.

2018-06-01

A technique to perform microscope self-calibration via micro laser line and soft computing algorithms is presented. In this technique, the microscope vision parameters are computed by means of soft computing algorithms based on laser line projection. To implement the self-calibration, a microscope vision system is constructed by means of a CCD camera and a 38 μm laser line. From this arrangement, the microscope vision parameters are represented via Bezier approximation networks, which are accomplished through the laser line position. In this procedure, a genetic algorithm determines the microscope vision parameters by means of laser line imaging. Also, the approximation networks compute the three-dimensional vision by means of the laser line position. Additionally, the soft computing algorithms re-calibrate the vision parameters when the microscope vision system is modified during the vision task. The proposed self-calibration improves accuracy of the traditional microscope calibration, which is accomplished via external references to the microscope system. The capability of the self-calibration based on soft computing algorithms is determined by means of the calibration accuracy and the micro-scale measurement error. This contribution is corroborated by an evaluation based on the accuracy of the traditional microscope calibration.

Increasing Student Understanding of Microscope Optics by Building and Testing the Limits of Simple, Hand-Made Model Microscopes†

PubMed Central

Drace, Kevin; Couch, Brett; Keeling, Patrick J.

2012-01-01

The ability to effectively use a microscope to observe microorganisms is a crucial skill required for many disciplines within biology, especially general microbiology and cell biology. A basic understanding of the optical properties of light microscopes is required for students to use microscopes effectively, but this subject can also be a challenge to make personally interesting to students. To explore basic optical principles of magnification and resolving power in a more engaging and hands-on fashion, students constructed handmade lenses and microscopes based on Antony van Leeuwenhoek’s design using simple materials—paper, staples, glass, and adhesive putty. Students determined the power of their lenses using a green laser pointer to magnify a copper grid of known size, which also allowed students to examine variables affecting the power and resolution of a lens such as diameter, working distance, and wavelength of light. To assess the effectiveness of the laboratory’s learning objectives, four sections of a general microbiology course were given a brief pre-activity assessment quiz to determine their background knowledge on the subject. One week after the laboratory activity, students were given the same quiz (unannounced) under similar conditions. Students showed significant gains in their understanding of microscope optics. PMID:23653781