PScan 1.0: flexible software framework for polygon based multiphoton microscopy

NASA Astrophysics Data System (ADS)

Li, Yongxiao; Lee, Woei Ming

2016-12-01

Multiphoton laser scanning microscopes exhibit highly localized nonlinear optical excitation and are powerful instruments for in-vivo deep tissue imaging. Customized multiphoton microscopy has a significantly superior performance for in-vivo imaging because of precise control over the scanning and detection system. To date, there have been several flexible software platforms catered to custom built microscopy systems i.e. ScanImage, HelioScan, MicroManager, that perform at imaging speeds of 30-100fps. In this paper, we describe a flexible software framework for high speed imaging systems capable of operating from 5 fps to 1600 fps. The software is based on the MATLAB image processing toolbox. It has the capability to communicate directly with a high performing imaging card (Matrox Solios eA/XA), thus retaining high speed acquisition. The program is also designed to communicate with LabVIEW and Fiji for instrument control and image processing. Pscan 1.0 can handle high imaging rates and contains sufficient flexibility for users to adapt to their high speed imaging systems.

Image correlation microscopy for uniform illumination.

PubMed

Gaborski, T R; Sealander, M N; Ehrenberg, M; Waugh, R E; McGrath, J L

2010-01-01

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

Liquid scanning transmission electron microscopy: imaging protein complexes in their native environment in whole eukaryotic cells.

PubMed

Peckys, Diana B; de Jonge, Niels

2014-04-01

Scanning transmission electron microscopy (STEM) of specimens in liquid, so-called Liquid STEM, is capable of imaging the individual subunits of macromolecular complexes in whole eukaryotic cells in liquid. This paper discusses this new microscopy modality within the context of state-of-the-art microscopy of cells. The principle of operation and equations for the resolution are described. The obtained images are different from those acquired with standard transmission electron microscopy showing the cellular ultrastructure. Instead, contrast is obtained on specific labels. Images can be recorded in two ways, either via STEM at 200 keV electron beam energy using a microfluidic chamber enclosing the cells, or via environmental scanning electron microscopy at 30 keV of cells in a wet environment. The first series of experiments involved the epidermal growth factor receptor labeled with gold nanoparticles. The labels were imaged in whole fixed cells with nanometer resolution. Since the cells can be kept alive in the microfluidic chamber, it is also feasible to detect the labels in unfixed, live cells. The rapid sample preparation and imaging allows studies of multiple whole cells.

Image scanning fluorescence emission difference microscopy based on a detector array.

PubMed

Li, Y; Liu, S; Liu, D; Sun, S; Kuang, C; Ding, Z; Liu, X

2017-06-01

We propose a novel imaging method that enables the enhancement of three-dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

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

PubMed Central

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

2017-01-01

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

On the Progress of Scanning Transmission Electron Microscopy (STEM) Imaging in a Scanning Electron Microscope.

PubMed

Sun, Cheng; Müller, Erich; Meffert, Matthias; Gerthsen, Dagmar

2018-04-01

Transmission electron microscopy (TEM) with low-energy electrons has been recognized as an important addition to the family of electron microscopies as it may avoid knock-on damage and increase the contrast of weakly scattering objects. Scanning electron microscopes (SEMs) are well suited for low-energy electron microscopy with maximum electron energies of 30 keV, but they are mainly used for topography imaging of bulk samples. Implementation of a scanning transmission electron microscopy (STEM) detector and a charge-coupled-device camera for the acquisition of on-axis transmission electron diffraction (TED) patterns, in combination with recent resolution improvements, make SEMs highly interesting for structure analysis of some electron-transparent specimens which are traditionally investigated by TEM. A new aspect is correlative SEM, STEM, and TED imaging from the same specimen region in a SEM which leads to a wealth of information. Simultaneous image acquisition gives information on surface topography, inner structure including crystal defects and qualitative material contrast. Lattice-fringe resolution is obtained in bright-field STEM imaging. The benefits of correlative SEM/STEM/TED imaging in a SEM are exemplified by structure analyses from representative sample classes such as nanoparticulates and bulk materials.

Three-dimensional nanoscale imaging by plasmonic Brownian microscopy

NASA Astrophysics Data System (ADS)

Labno, Anna; Gladden, Christopher; Kim, Jeongmin; Lu, Dylan; Yin, Xiaobo; Wang, Yuan; Liu, Zhaowei; Zhang, Xiang

2017-12-01

Three-dimensional (3D) imaging at the nanoscale is a key to understanding of nanomaterials and complex systems. While scanning probe microscopy (SPM) has been the workhorse of nanoscale metrology, its slow scanning speed by a single probe tip can limit the application of SPM to wide-field imaging of 3D complex nanostructures. Both electron microscopy and optical tomography allow 3D imaging, but are limited to the use in vacuum environment due to electron scattering and to optical resolution in micron scales, respectively. Here we demonstrate plasmonic Brownian microscopy (PBM) as a way to improve the imaging speed of SPM. Unlike photonic force microscopy where a single trapped particle is used for a serial scanning, PBM utilizes a massive number of plasmonic nanoparticles (NPs) under Brownian diffusion in solution to scan in parallel around the unlabeled sample object. The motion of NPs under an evanescent field is three-dimensionally localized to reconstruct the super-resolution topology of 3D dielectric objects. Our method allows high throughput imaging of complex 3D structures over a large field of view, even with internal structures such as cavities that cannot be accessed by conventional mechanical tips in SPM.

Scanning superlens microscopy for non-invasive large field-of-view visible light nanoscale imaging

NASA Astrophysics Data System (ADS)

Wang, Feifei; Liu, Lianqing; Yu, Haibo; Wen, Yangdong; Yu, Peng; Liu, Zhu; Wang, Yuechao; Li, Wen Jung

2016-12-01

Nanoscale correlation of structural information acquisition with specific-molecule identification provides new insight for studying rare subcellular events. To achieve this correlation, scanning electron microscopy has been combined with super-resolution fluorescent microscopy, despite its destructivity when acquiring biological structure information. Here we propose time-efficient non-invasive microsphere-based scanning superlens microscopy that enables the large-area observation of live-cell morphology or sub-membrane structures with sub-diffraction-limited resolution and is demonstrated by observing biological and non-biological objects. This microscopy operates in both non-invasive and contact modes with ~200 times the acquisition efficiency of atomic force microscopy, which is achieved by replacing the point of an atomic force microscope tip with an imaging area of microspheres and stitching the areas recorded during scanning, enabling sub-diffraction-limited resolution. Our method marks a possible path to non-invasive cell imaging and simultaneous tracking of specific molecules with nanoscale resolution, facilitating the study of subcellular events over a total cell period.

Fast and reliable method of conductive carbon nanotube-probe fabrication for scanning probe microscopy

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

Dremov, Vyacheslav, E-mail: dremov@issp.ac.ru; Fedorov, Pavel; Grebenko, Artem

2015-05-15

We demonstrate the procedure of scanning probe microscopy (SPM) conductive probe fabrication with a single multi-walled carbon nanotube (MWNT) on a silicon cantilever pyramid. The nanotube bundle reliably attached to the metal-covered pyramid is formed using dielectrophoresis technique from the MWNT suspension. It is shown that the dimpled aluminum sample can be used both for shortening/modification of the nanotube bundle by applying pulse voltage between the probe and the sample and for controlling the probe shape via atomic force microscopy imaging the sample. Carbon nanotube attached to cantilever covered with noble metal is suitable for SPM imaging in such modulationmore » regimes as capacitance contrast microscopy, Kelvin probe microscopy, and scanning gate microscopy. The majority of such probes are conductive with conductivity not degrading within hours of SPM imaging.« less

Fluorescence microscopy.

PubMed

Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

2014-10-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

Fluorescence Microscopy

PubMed Central

Sanderson, Michael J.; Smith, Ian; Parker, Ian; Bootman, Martin D.

2016-01-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. PMID:25275114

Environmental scanning electron microscopy in cell biology.

PubMed

McGregor, J E; Staniewicz, L T L; Guthrie Neé Kirk, S E; Donald, A M

2013-01-01

Environmental scanning electron microscopy (ESEM) (1) is an imaging technique which allows hydrated, insulating samples to be imaged under an electron beam. The resolution afforded by this technique is higher than conventional optical microscopy but lower than conventional scanning electron microscopy (CSEM). The major advantage of the technique is the minimal sample preparation needed, making ESEM quick to use and the images less susceptible to the artifacts that the extensive sample preparation usually required for CSEM may introduce. Careful manipulation of both the humidity in the microscope chamber and the beam energy are nevertheless essential to prevent dehydration and beam damage artifacts. In some circumstances it is possible to image live cells in the ESEM (2).In the following sections we introduce the fundamental principles of ESEM imaging before presenting imaging protocols for plant epidermis, mammalian cells, and bacteria. In the first two cases samples are imaged using the secondary electron (topographic) signal, whereas a transmission technique is employed to image bacteria.

Integrated one- and two-photon scanned oblique plane illumination (SOPi) microscopy for rapid volumetric imaging

NASA Astrophysics Data System (ADS)

Kumar, Manish; Kishore, Sandeep; Nasenbeny, Jordan; McLean, David L.; Kozorovitskiy, Yevgenia

2018-05-01

Versatile, sterically accessible imaging systems capable of in vivo rapid volumetric functional and structural imaging deep in the brain continue to be a limiting factor in neuroscience research. Towards overcoming this obstacle, we present integrated one- and two-photon scanned oblique plane illumination (SOPi) microscopy which uses a single front-facing microscope objective to provide light-sheet scanning based rapid volumetric imaging capability at subcellular resolution. Our planar scan-mirror based optimized light-sheet architecture allows for non-distorted scanning of volume samples, simplifying accurate reconstruction of the imaged volume. Integration of both one-photon (1P) and two-photon (2P) light-sheet microscopy in the same system allows for easy selection between rapid volumetric imaging and higher resolution imaging in scattering media. Using SOPi, we demonstrate deep, large volume imaging capability inside scattering mouse brain sections and rapid imaging speeds up to 10 volumes per second in zebrafish larvae expressing genetically encoded fluorescent proteins GFP or GCaMP6s. SOPi flexibility and steric access makes it adaptable for numerous imaging applications and broadly compatible with orthogonal techniques for actuating or interrogating neuronal structure and activity.

Integrated one- and two-photon scanned oblique plane illumination (SOPi) microscopy for rapid volumetric imaging.

PubMed

Kumar, Manish; Kishore, Sandeep; Nasenbeny, Jordan; McLean, David L; Kozorovitskiy, Yevgenia

2018-05-14

Versatile, sterically accessible imaging systems capable of in vivo rapid volumetric functional and structural imaging deep in the brain continue to be a limiting factor in neuroscience research. Towards overcoming this obstacle, we present integrated one- and two-photon scanned oblique plane illumination (SOPi, /sōpī/) microscopy which uses a single front-facing microscope objective to provide light-sheet scanning based rapid volumetric imaging capability at subcellular resolution. Our planar scan-mirror based optimized light-sheet architecture allows for non-distorted scanning of volume samples, simplifying accurate reconstruction of the imaged volume. Integration of both one-photon (1P) and two-photon (2P) light-sheet microscopy in the same system allows for easy selection between rapid volumetric imaging and higher resolution imaging in scattering media. Using SOPi, we demonstrate deep, large volume imaging capability inside scattering mouse brain sections and rapid imaging speeds up to 10 volumes per second in zebrafish larvae expressing genetically encoded fluorescent proteins GFP or GCaMP6s. SOPi's flexibility and steric access makes it adaptable for numerous imaging applications and broadly compatible with orthogonal techniques for actuating or interrogating neuronal structure and activity.

Adaptive and robust statistical methods for processing near-field scanning microwave microscopy images.

PubMed

Coakley, K J; Imtiaz, A; Wallis, T M; Weber, J C; Berweger, S; Kabos, P

2015-03-01

Near-field scanning microwave microscopy offers great potential to facilitate characterization, development and modeling of materials. By acquiring microwave images at multiple frequencies and amplitudes (along with the other modalities) one can study material and device physics at different lateral and depth scales. Images are typically noisy and contaminated by artifacts that can vary from scan line to scan line and planar-like trends due to sample tilt errors. Here, we level images based on an estimate of a smooth 2-d trend determined with a robust implementation of a local regression method. In this robust approach, features and outliers which are not due to the trend are automatically downweighted. We denoise images with the Adaptive Weights Smoothing method. This method smooths out additive noise while preserving edge-like features in images. We demonstrate the feasibility of our methods on topography images and microwave |S11| images. For one challenging test case, we demonstrate that our method outperforms alternative methods from the scanning probe microscopy data analysis software package Gwyddion. Our methods should be useful for massive image data sets where manual selection of landmarks or image subsets by a user is impractical. Published by Elsevier B.V.

Correcting nonlinear drift distortion of scanning probe and scanning transmission electron microscopies from image pairs with orthogonal scan directions

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

Ophus, Colin; Ciston, Jim; Nelson, Chris T.

Unwanted motion of the probe with respect to the sample is a ubiquitous problem in scanning probe and scanning transmission electron microscopies, causing both linear and nonlinear artifacts in experimental images. We have designed a procedure to correct these artifacts by using orthogonal scan pairs to align each measurement line-by-line along the slow scan direction, by fitting contrast variation along the lines. We demonstrate the accuracy of our algorithm on both synthetic and experimental data and provide an implementation of our method.

Correcting nonlinear drift distortion of scanning probe and scanning transmission electron microscopies from image pairs with orthogonal scan directions

DOE PAGES

Ophus, Colin; Ciston, Jim; Nelson, Chris T.

2015-12-10

Unwanted motion of the probe with respect to the sample is a ubiquitous problem in scanning probe and scanning transmission electron microscopies, causing both linear and nonlinear artifacts in experimental images. We have designed a procedure to correct these artifacts by using orthogonal scan pairs to align each measurement line-by-line along the slow scan direction, by fitting contrast variation along the lines. We demonstrate the accuracy of our algorithm on both synthetic and experimental data and provide an implementation of our method.

Volumetric structured illumination microscopy enabled by tunable focus lens (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hinsdale, Taylor; Malik, Bilal; Olsovsky, Cory; Jo, Javier A.; Maitland, Kristen C.

2016-03-01

We present a volumetric imaging method for biological tissue that is free of mechanically scanning components. The optical sectioning in the system is obtained by structured illumination microscopy (SIM) with the depth of focus being varied by the use of an electronic tunable-focus lens (ETL). The performance of the axial scanning mechanism was evaluated and characterized in conjunction with SIM to ensure volumetric images could be recorded and reconstructed without significant losses in optical section thickness and lateral resolution over the full desired scan range. It was demonstrated that sub-cellular image resolutions were obtainable in both microsphere films and in ex vivo oral mucosa, spanning multiple cell layers, without significant losses in image quality. The mechanism proposed here has the ability to be integrated into any wide-field microscopy system to convert it into a three-dimensional imaging platform without the need for axial scanning of the sample or imaging optics. The ability to axially scan independent of mechanical movement also provides the opportunity for the development of endoscopic systems which can create volumetric images of tissue in vivo.

System and method for compressive scanning electron microscopy

DOEpatents

Reed, Bryan W

2015-01-13

A scanning transmission electron microscopy (STEM) system is disclosed. The system may make use of an electron beam scanning system configured to generate a plurality of electron beam scans over substantially an entire sample, with each scan varying in electron-illumination intensity over a course of the scan. A signal acquisition system may be used for obtaining at least one of an image, a diffraction pattern, or a spectrum from the scans, the image, diffraction pattern, or spectrum representing only information from at least one of a select subplurality or linear combination of all pixel locations comprising the image. A dataset may be produced from the information. A subsystem may be used for mathematically analyzing the dataset to predict actual information that would have been produced by each pixel location of the image.

Qualitative and quantitative interpretation of SEM image using digital image processing.

PubMed

Saladra, Dawid; Kopernik, Magdalena

2016-10-01

The aim of the this study is improvement of qualitative and quantitative analysis of scanning electron microscope micrographs by development of computer program, which enables automatic crack analysis of scanning electron microscopy (SEM) micrographs. Micromechanical tests of pneumatic ventricular assist devices result in a large number of micrographs. Therefore, the analysis must be automatic. Tests for athrombogenic titanium nitride/gold coatings deposited on polymeric substrates (Bionate II) are performed. These tests include microshear, microtension and fatigue analysis. Anisotropic surface defects observed in the SEM micrographs require support for qualitative and quantitative interpretation. Improvement of qualitative analysis of scanning electron microscope images was achieved by a set of computational tools that includes binarization, simplified expanding, expanding, simple image statistic thresholding, the filters Laplacian 1, and Laplacian 2, Otsu and reverse binarization. Several modifications of the known image processing techniques and combinations of the selected image processing techniques were applied. The introduced quantitative analysis of digital scanning electron microscope images enables computation of stereological parameters such as area, crack angle, crack length, and total crack length per unit area. This study also compares the functionality of the developed computer program of digital image processing with existing applications. The described pre- and postprocessing may be helpful in scanning electron microscopy and transmission electron microscopy surface investigations. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Scanning Transmission Electron Microscopy | Materials Science | NREL

Science.gov Websites

mode by collecting the EDS and EELS signals point-by-point as one scans the electron probe across the . Examples of Scanning Transmission Electron Microscopy Capabilities Z-contrast image microphoto taken by

New developments in electron microscopy for serial image acquisition of neuronal profiles.

PubMed

Kubota, Yoshiyuki

2015-02-01

Recent developments in electron microscopy largely automate the continuous acquisition of serial electron micrographs (EMGs), previously achieved by laborious manual serial ultrathin sectioning using an ultramicrotome and ultrastructural image capture process with transmission electron microscopy. The new systems cut thin sections and capture serial EMGs automatically, allowing for acquisition of large data sets in a reasonably short time. The new methods are focused ion beam/scanning electron microscopy, ultramicrotome/serial block-face scanning electron microscopy, automated tape-collection ultramicrotome/scanning electron microscopy and transmission electron microscope camera array. In this review, their positive and negative aspects are discussed. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

High-resolution photoluminescence electro-modulation microscopy by scanning lock-in

NASA Astrophysics Data System (ADS)

Koopman, W.; Muccini, M.; Toffanin, S.

2018-04-01

Morphological inhomogeneities and structural defects in organic semiconductors crucially determine the charge accumulation and lateral transport in organic thin-film transistors. Photoluminescence Electro-Modulation (PLEM) microscopy is a laser-scanning microscopy technique that relies on the modulation of the thin-film fluorescence in the presence of charge-carriers to image the spatial distribution of charges within the active organic semiconductor. Here, we present a lock-in scheme based on a scanning beam approach for increasing the PLEM microscopy resolution and contrast. The charge density in the device is modulated by a sinusoidal electrical signal, phase-locked to the scanning beam of the excitation laser. The lock-in detection scheme is achieved by acquiring a series of images with different phases between the beam scan and the electrical modulation. Application of high resolution PLEM to an organic transistor in accumulation mode demonstrates its potential to image local variations in the charge accumulation. A diffraction-limited precision of sub-300 nm and a signal to noise ratio of 21.4 dB could be achieved.

Imaging of high-angle annular dark-field scanning transmission electron microscopy and observations of GaN-based violet laser diodes.

PubMed

Shiojiri, M; Saijo, H

2006-09-01

The first part of this paper is devoted to physics, to explain high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) imaging and to interpret why HAADF-STEM imaging is incoherent, instructing a strict definition of interference and coherence of electron waves. Next, we present our recent investigations of InGaN/GaN multiple quantum wells and AlGaN/GaN strained-layer superlattice claddings in GaN-based violet laser diodes, which have been performed by HAADF-STEM and high-resolution field-emission gun scanning electron microscopy.

Remote focusing for programmable multi-layer differential multiphoton microscopy

PubMed Central

Hoover, Erich E.; Young, Michael D.; Chandler, Eric V.; Luo, Anding; Field, Jeffrey J.; Sheetz, Kraig E.; Sylvester, Anne W.; Squier, Jeff A.

2010-01-01

We present the application of remote focusing to multiphoton laser scanning microscopy and utilize this technology to demonstrate simultaneous, programmable multi-layer imaging. Remote focusing is used to independently control the axial location of multiple focal planes that can be simultaneously imaged with single element detection. This facilitates volumetric multiphoton imaging in scattering specimens and can be practically scaled to a large number of focal planes. Further, it is demonstrated that the remote focusing control can be synchronized with the lateral scan directions, enabling imaging in orthogonal scan planes. PMID:21326641

Scanning Tunneling Microscopy | Materials Science | NREL

Science.gov Websites

of Scanning Tunneling Microscopy Capabilities Two-dimensional STM image 2D STM image of Si(111) 7×7 clusters along a fault boundary Occupied-state STM image taken on a Si(111)7×7 surface, showing two 7×7 tip at each point, an image of the sample surface is generated (topographic image). For very flat

Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid.

PubMed

Dukes, Madeline J; Peckys, Diana B; de Jonge, Niels

2010-07-27

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.

Correlative Fluorescence Microscopy and Scanning Transmission Electron Microscopy of Quantum Dot Labeled Proteins in Whole Cells in Liquid

PubMed Central

Dukes, Madeline J.; Peckys, Diana B.; de Jonge, Niels

2010-01-01

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7 × 12 nm were visible in a 5 μm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs. PMID:20550177

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.

Artifact mitigation of ptychography integrated with on-the-fly scanning probe microscopy

DOE PAGES

Huang, Xiaojing; Yan, Hanfei; Ge, Mingyuan; ...

2017-07-11

In this paper, we report our experiences with conducting ptychography simultaneously with the X-ray fluorescence measurement using the on-the-fly mode for efficient multi-modality imaging. We demonstrate that the periodic artifact inherent to the raster scan pattern can be mitigated using a sufficiently fine scan step size to provide an overlap ratio of >70%. This allows us to obtain transmitted phase contrast images with enhanced spatial resolution from ptychography while maintaining the fluorescence imaging with continuous-motion scans on pixelated grids. Lastly, this capability will greatly improve the competence and throughput of scanning probe X-ray microscopy.

Spatial Resolution in Scanning Electron Microscopy and Scanning Transmission Electron Microscopy Without a Specimen Vacuum Chamber.

PubMed

Nguyen, Kayla X; Holtz, Megan E; Richmond-Decker, Justin; Muller, David A

2016-08-01

A long-standing goal of electron microscopy has been the high-resolution characterization of specimens in their native environment. However, electron optics require high vacuum to maintain an unscattered and focused probe, a challenge for specimens requiring atmospheric or liquid environments. Here, we use an electron-transparent window at the base of a scanning electron microscope's objective lens to separate column vacuum from the specimen, enabling imaging under ambient conditions, without a specimen vacuum chamber. We demonstrate in-air imaging of specimens at nanoscale resolution using backscattered scanning electron microscopy (airSEM) and scanning transmission electron microscopy. We explore resolution and contrast using Monte Carlo simulations and analytical models. We find that nanometer-scale resolution can be obtained at gas path lengths up to 400 μm, although contrast drops with increasing gas path length. As the electron-transparent window scatters considerably more than gas at our operating conditions, we observe that the densities and thicknesses of the electron-transparent window are the dominant limiting factors for image contrast at lower operating voltages. By enabling a variety of detector configurations, the airSEM is applicable to a wide range of environmental experiments including the imaging of hydrated biological specimens and in situ chemical and electrochemical processes.

Spatial Resolution in Scanning Electron Microscopy and Scanning Transmission Electron Microscopy Without a Specimen Vacuum Chamber

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

Nguyen, Kayla X.; Holtz, Megan E.; Richmond-Decker, Justin

2016-07-25

Abstract A long-standing goal of electron microscopy has been the high-resolution characterization of specimens in their native environment. However, electron optics require high vacuum to maintain an unscattered and focused probe, a challenge for specimens requiring atmospheric or liquid environments. Here, we use an electron-transparent window at the base of a scanning electron microscope’s objective lens to separate column vacuum from the specimen, enabling imaging under ambient conditions, without a specimen vacuum chamber. We demonstrate in-air imaging of specimens at nanoscale resolution using backscattered scanning electron microscopy (airSEM) and scanning transmission electron microscopy. We explore resolution and contrast using Montemore » Carlo simulations and analytical models. We find that nanometer-scale resolution can be obtained at gas path lengths up to 400μm, although contrast drops with increasing gas path length. As the electron-transparent window scatters considerably more than gas at our operating conditions, we observe that the densities and thicknesses of the electron-transparent window are the dominant limiting factors for image contrast at lower operating voltages. By enabling a variety of detector configurations, the airSEM is applicable to a wide range of environmental experiments including the imaging of hydrated biological specimens andin situchemical and electrochemical processes.« less

Three-dimensional imaging of adherent cells using FIB/SEM and STEM.

PubMed

Villinger, Clarissa; Schauflinger, Martin; Gregorius, Heiko; Kranz, Christine; Höhn, Katharina; Nafeey, Soufi; Walther, Paul

2014-01-01

In this chapter we describe three different approaches for three-dimensional imaging of electron microscopic samples: serial sectioning transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM) tomography, and focused ion beam/scanning electron microscopy (FIB/SEM) tomography. With these methods, relatively large volumes of resin-embedded biological structures can be analyzed at resolutions of a few nm within a reasonable expenditure of time. The traditional method is serial sectioning and imaging the same area in all sections. Another method is TEM tomography that involves tilting a section in the electron beam and then reconstruction of the volume by back projection of the images. When the scanning transmission (STEM) mode is used, thicker sections (up to 1 μm) can be analyzed. The third approach presented here is focused ion beam/scanning electron microscopy (FIB/SEM) tomography, in which a sample is repeatedly milled with a focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrary small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. We show that resolution of this approach is considerably improved when the secondary electron signal is used. However, the most important prerequisite for three-dimensional imaging is good specimen preparation. For all three imaging methods, cryo-fixed (high-pressure frozen) and freeze-substituted samples have been used.

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

PubMed

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

2014-03-01

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

Scalp imaging techniques

NASA Astrophysics Data System (ADS)

Otberg, Nina; Shapiro, Jerry; Lui, Harvey; Wu, Wen-Yu; Alzolibani, Abdullateef; Kang, Hoon; Richter, Heike; Lademann, Jürgen

2017-05-01

Scalp imaging techniques are necessary tools for the trichological practice and for visualization of permeation, penetration and absorption processes into and through the scalp and for the research on drug delivery and toxicology. The present letter reviews different scalp imaging techniques and discusses their utility. Moreover, two different studies on scalp imaging techniques are presented in this letter: (1) scalp imaging with phototrichograms in combination with laser scanning microscopy, and (2) follicular measurements with cyanoacrylate surface replicas and light microscopy in combination with laser scanning microscopy. The experiments compare different methods for the determination of hair density on the scalp and different follicular measures. An average terminal hair density of 132 hairs cm-2 was found in 6 Caucasian volunteers and 135 hairs cm-2 in 6 Asian volunteers. The area of the follicular orifices accounts to 16.3% of the skin surface on average measured with laser scanning microscopy images. The potential volume of the follicular infundibulum was calculated based on the laser scanning measurements and is found to be 4.63 mm3 per cm2 skin on average. The experiments show that hair follicles are quantitatively relevant pathways and potential reservoirs for topically applied drugs and cosmetics.

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

PubMed

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

2015-01-01

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

Imaging graphite in air by scanning tunneling microscopy - Role of the tip

NASA Technical Reports Server (NTRS)

Colton, R. J.; Baker, S. M.; Driscoll, R. J.; Youngquist, M. G.; Baldeschwieler, J. D.; Kaiser, W. J.

1988-01-01

Atomically resolved images of highly oriented pyrolytic graphite (HOPG) in air at point contact have been obtained. Direct contact between tip and sample or contact through a contamination layer provides a conduction mechanism in addition to the exponential tunneling mechanism responsible for scanning tunneling microscopy (STM) imaging. Current-voltage (I-V) spectra were obtained while scanning in the current imaging mode with the feedback circuit interrupted in order to study the graphite imaging mechanism. Multiple tunneling tips are probably responsible for images without the expected hexagonal or trigonal symmetry. The observations indicate that the use of HOPG for testing and calibration of STM instrumentation may be misleading.

Direct visualization of lithium via annular bright field scanning transmission electron microscopy: a review.

PubMed

Findlay, Scott David; Huang, Rong; Ishikawa, Ryo; Shibata, Naoya; Ikuhara, Yuichi

2017-02-08

Annular bright field (ABF) scanning transmission electron microscopy has proven able to directly image lithium columns within crystalline environments, offering much insight into the structure and properties of lithium-ion battery materials. We summarize the image formation mechanisms underpinning ABF imaging, review the experimental application of this technique to imaging lithium in materials and overview the conditions that help maximize the visibility of lithium columns. © The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Image scanning microscopy using a SPAD detector array (Conference Presentation)

NASA Astrophysics Data System (ADS)

Castello, Marco; Tortarolo, Giorgio; Buttafava, Mauro; Tosi, Alberto; Sheppard, Colin J. R.; Diaspro, Alberto; Vicidomini, Giuseppe

2017-02-01

The use of an array of detectors can help overcoming the traditional limitation of confocal microscopy: the compromise between signal and theoretical resolution. Each element independently records a view of the sample and the final image can be reconstructed by pixel reassignment or by inverse filtering (e.g. deconvolution). In this work, we used a SPAD array of 25 detectors specifically designed for this goal and our scanning microscopy control system (Carma) to acquire the partial images and to perform online image processing. Further work will be devoted to optimize the image reconstruction step and to improve the fill-factor of the detector.

Scanning capacitance microscopy of ErAs nanoparticles embedded in GaAs pn junctions

NASA Astrophysics Data System (ADS)

Park, K. W.; Nair, H. P.; Crook, A. M.; Bank, S. R.; Yu, E. T.

2011-09-01

Scanning capacitance microscopy is used to characterize the electronic properties of ErAs nanoparticles embedded in GaAs pn junctions grown by molecular beam epitaxy. Voltage-dependent capacitance images reveal localized variations in subsurface electronic structure near buried ErAs nanoparticles at lateral length scales of 20-30 nm. Numerical modeling indicates that these variations arise from inhomogeneities in charge modulation due to Fermi level pinning behavior associated with the embedded ErAs nanoparticles. Statistical analysis of image data yields an average particle radius of 6-8 nm—well below the direct resolution limit in scanning capacitance microscopy but discernible via analysis of patterns in nanoscale capacitance images.

Signal-to-noise ratio estimation on SEM images using cubic spline interpolation with Savitzky-Golay smoothing.

PubMed

Sim, K S; Kiani, M A; Nia, M E; Tso, C P

2014-01-01

A new technique based on cubic spline interpolation with Savitzky-Golay noise reduction filtering is designed to estimate signal-to-noise ratio of scanning electron microscopy (SEM) images. This approach is found to present better result when compared with two existing techniques: nearest neighbourhood and first-order interpolation. When applied to evaluate the quality of SEM images, noise can be eliminated efficiently with optimal choice of scan rate from real-time SEM images, without generating corruption or increasing scanning time. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

Insights into radiation damage from atomic resolution scanning transmission electron microscopy imaging of mono-layer CuPcCl16 films on graphene.

PubMed

Mittelberger, Andreas; Kramberger, Christian; Meyer, Jannik C

2018-03-19

Atomically resolved images of monolayer organic crystals have only been obtained with scanning probe methods so far. On the one hand, they are usually prepared on surfaces of bulk materials, which are not accessible by (scanning) transmission electron microscopy. On the other hand, the critical electron dose of a monolayer organic crystal is orders of magnitudes lower than the one for bulk crystals, making (scanning) transmission electron microscopy characterization very challenging. In this work we present an atomically resolved study on the dynamics of a monolayer CuPcCl 16 crystal under the electron beam as well as an image of the undamaged molecules obtained by low-dose electron microscopy. The results show the dynamics and the radiation damage mechanisms in the 2D layer of this material, complementing what has been found for bulk crystals in earlier studies. Furthermore, being able to image the undamaged molecular crystal allows the characterization of new composites consisting of 2D materials and organic molecules.

Seeing through walls at the nanoscale: Microwave microscopy of enclosed objects and processes in liquids

DOE PAGES

Velmurugan, Jeyavel; Kalinin, Sergei V.; Kolmakov, Andrei; ...

2016-02-11

Here, noninvasive in situ nanoscale imaging in liquid environments is a current imperative in the analysis of delicate biomedical objects and electrochemical processes at reactive liquid–solid interfaces. Microwaves of a few gigahertz frequencies offer photons with energies of ≈10 μeV, which can affect neither electronic states nor chemical bonds in condensed matter. Here, we describe an implementation of scanning near-field microwave microscopy for imaging in liquids using ultrathin molecular impermeable membranes separating scanning probes from samples enclosed in environmental cells. We imaged a model electroplating reaction as well as individual live cells. Through a side-by-side comparison of the microwave imagingmore » with scanning electron microscopy, we demonstrate the advantage of microwaves for artifact-free imaging.« less

Multidepth imaging by chromatic dispersion confocal microscopy

NASA Astrophysics Data System (ADS)

Olsovsky, Cory A.; Shelton, Ryan L.; Saldua, Meagan A.; Carrasco-Zevallos, Oscar; Applegate, Brian E.; Maitland, Kristen C.

2012-03-01

Confocal microscopy has shown potential as an imaging technique to detect precancer. Imaging cellular features throughout the depth of epithelial tissue may provide useful information for diagnosis. However, the current in vivo axial scanning techniques for confocal microscopy are cumbersome, time-consuming, and restrictive when attempting to reconstruct volumetric images acquired in breathing patients. Chromatic dispersion confocal microscopy (CDCM) exploits severe longitudinal chromatic aberration in the system to axially disperse light from a broadband source and, ultimately, spectrally encode high resolution images along the depth of the object. Hyperchromat lenses are designed to have severe and linear longitudinal chromatic aberration, but have not yet been used in confocal microscopy. We use a hyperchromat lens in a stage scanning confocal microscope to demonstrate the capability to simultaneously capture information at multiple depths without mechanical scanning. A photonic crystal fiber pumped with a 830nm wavelength Ti:Sapphire laser was used as a supercontinuum source, and a spectrometer was used as the detector. The chromatic aberration and magnification in the system give a focal shift of 140μm after the objective lens and an axial resolution of 5.2-7.6μm over the wavelength range from 585nm to 830nm. A 400x400x140μm3 volume of pig cheek epithelium was imaged in a single X-Y scan. Nuclei can be seen at several depths within the epithelium. The capability of this technique to achieve simultaneous high resolution confocal imaging at multiple depths may reduce imaging time and motion artifacts and enable volumetric reconstruction of in vivo confocal images of the epithelium.

Theoretical study of carbon-based tips for scanning tunnelling microscopy.

PubMed

González, C; Abad, E; Dappe, Y J; Cuevas, J C

2016-03-11

Motivated by recent experiments, we present here a detailed theoretical analysis of the use of carbon-based conductive tips in scanning tunnelling microscopy. In particular, we employ ab initio methods based on density functional theory to explore a graphitic, an amorphous carbon and two diamond-like tips for imaging with a scanning tunnelling microscope (STM), and we compare them with standard metallic tips made of gold and tungsten. We investigate the performance of these tips in terms of the corrugation of the STM images acquired when scanning a single graphene sheet. Moreover, we analyse the impact of the tip-sample distance and show that it plays a fundamental role in the resolution and symmetry of the STM images. We also explore in depth how the adsorption of single atoms and molecules in the tip apexes modifies the STM images and demonstrate that, in general, it leads to an improved image resolution. The ensemble of our results provides strong evidence that carbon-based tips can significantly improve the resolution of STM images, as compared to more standard metallic tips, which may open a new line of research in scanning tunnelling microscopy.

Atomic force microscopy of biological samples

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

Doktycz, Mitchel John

2010-01-01

The ability to evaluate structural-functional relationships in real time has allowed scanning probe microscopy (SPM) to assume a prominent role in post genomic biological research. In this mini-review, we highlight the development of imaging and ancillary techniques that have allowed SPM to permeate many key areas of contemporary research. We begin by examining the invention of the scanning tunneling microscope (STM) by Binnig and Rohrer in 1982 and discuss how it served to team biologists with physicists to integrate high-resolution microscopy into biological science. We point to the problems of imaging nonconductive biological samples with the STM and relate howmore » this led to the evolution of the atomic force microscope (AFM) developed by Binnig, Quate, and Gerber, in 1986. Commercialization in the late 1980s established SPM as a powerful research tool in the biological research community. Contact mode AFM imaging was soon complemented by the development of non-contact imaging modes. These non-contact modes eventually became the primary focus for further new applications including the development of fast scanning methods. The extreme sensitivity of the AFM cantilever was recognized and has been developed into applications for measuring forces required for indenting biological surfaces and breaking bonds between biomolecules. Further functional augmentation to the cantilever tip allowed development of new and emerging techniques including scanning ion-conductance microscopy (SICM), scanning electrochemical microscope (SECM), Kelvin force microscopy (KFM) and scanning near field ultrasonic holography (SNFUH).« less

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.

Correlative imaging of biological tissues with apertureless scanning near-field optical microscopy and confocal laser scanning microscopy

PubMed Central

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

2017-01-01

Apertureless scanning near-field optical microscopy (ASNOM) has attracted considerable interest over the past years as a result of its valuable contrast mechanisms and capabilities for optical resolutions in the nanoscale range. However, at this moment the intersections between ASNOM and the realm of bioimaging are scarce, mainly due to data interpretation difficulties linked to the limited body of work performed so far in this field and hence the reduced volume of supporting information. We propose an imaging approach that holds significant potential for alleviating this issue, consisting of correlative imaging of biological specimens using a multimodal system that incorporates ASNOM and confocal laser scanning microscopy (CLSM), which allows placing near-field data into a well understood context of anatomical relevance. We demonstrate this approach on zebrafish retinal tissue. The proposed method holds important implications for the in-depth understanding of biological items through the prism of ASNOM and CLSM data complementarity. PMID:29296474

Resolution enhancement in a double-helix phase engineered scanning microscope (RESCH microscope) (Presentation Recording)

NASA Astrophysics Data System (ADS)

Jesacher, Alexander; Ritsch-Marte, Monika; Piestun, Rafael

2015-08-01

Recently we introduced RESCH microscopy [1] - a scanning microscope that allows slightly refocusing the sample after the acquisition has been performed, solely by performing appropriate data post-processing. The microscope features a double-helix phase-engineered emission point spread function in combination with camera-based detection. Based on the principle of transverse resolution enhancement in Image Scanning Microscopy [2,3], we demonstrate similar resolution improvement in RESCH. Furthermore, we outline a pathway for how the collected 3D sample information can be used to construct sharper optical sections. [1] A. Jesacher, M. Ritsch-Marte and R. Piestun, accepted for Optica. [2] C.J.R. Sheppard, "Super-resolution in Confocal imaging," Optik, 80, 53-54 (1988). [3] C.B. Müller and J. Enderlein "Image Scanning Microscopy," Phys. Rev. Lett. 104, 198101 (2010).

Re-scan confocal microscopy: scanning twice for better resolution.

PubMed

De Luca, Giulia M R; Breedijk, Ronald M P; Brandt, Rick A J; Zeelenberg, Christiaan H C; de Jong, Babette E; Timmermans, Wendy; Azar, Leila Nahidi; Hoebe, Ron A; Stallinga, Sjoerd; Manders, Erik M M

2013-01-01

We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required.

X-ray ptychographic and fluorescence microscopy of frozen-hydrated cells using continuous scanning

DOE PAGES

Deng, Junjing; Vine, David J.; Chen, Si; ...

2017-03-27

X-ray microscopy can be used to image whole, unsectioned cells in their native hydrated state. It complements the higher resolution of electron microscopy for submicrometer thick specimens, and the molecule-specific imaging capabilites of fluorescence light microscopy. We describe here the first use of fast, continuous x-ray scanning of frozen hydrated cells for simultaneous sub-20 nm resolution ptychographic transmission imaging with high contrast, and sub-100 nm resolution deconvolved x-ray fluorescence imaging of diffusible and bound ions at native concentrations, without the need to add specific labels. Here, by working with cells that have been rapidly frozen without the use of chemicalmore » fixatives, and imaging them under cryogenic conditions, we are able to obtain images with well preserved structural and chemical composition, and sufficient stability against radiation damage to allow for multiple images to be obtained with no observable change.« less

Invited Review Article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy

PubMed Central

Carriles, Ramón; Schafer, Dawn N.; Sheetz, Kraig E.; Field, Jeffrey J.; Cisek, Richard; Barzda, Virginijus; Sylvester, Anne W.; Squier, Jeffrey A.

2009-01-01

We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences. PMID:19725639

Multimodal backside imaging of a microcontroller using confocal laser scanning and optical-beam-induced current imaging

NASA Astrophysics Data System (ADS)

Finkeldey, Markus; Göring, Lena; Schellenberg, Falk; Brenner, Carsten; Gerhardt, Nils C.; Hofmann, Martin

2017-02-01

Microscopy imaging with a single technology is usually restricted to a single contrast mechanism. Multimodal imaging is a promising technique to improve the structural information that could be obtained about a device under test (DUT). Due to the different contrast mechanisms of laser scanning microscopy (LSM), confocal laser scanning microscopy (CLSM) and optical beam induced current microscopy (OBICM), a combination could improve the detection of structures in integrated circuits (ICs) and helps to reveal their layout. While OBIC imaging is sensitive to the changes between differently doped areas and to semiconductor-metal transitions, CLSM imaging is mostly sensitive to changes in absorption and reflection. In this work we present the implementation of OBIC imaging into a CLSM. We show first results using industry standard Atmel microcontrollers (MCUs) with a feature size of about 250nm as DUTs. Analyzing these types of microcontrollers helps to improve in the field of side-channel attacks to find hardware Trojans, possible spots for laser fault attacks and for reverse engineering. For the experimental results the DUT is placed on a custom circuit board that allows us to measure the current while imaging it in our in-house built stage scanning microscope using a near infrared (NIR) laser diode as light source. The DUT is thinned and polished, allowing backside imaging through the Si-substrate. We demonstrate the possibilities using this optical setup by evaluating OBIC, LSM and CLSM images above and below the threshold of the laser source.

Reflection imaging of China ink-perfused brain vasculature using confocal laser-scanning microscopy after clarification of brain tissue by the Spalteholz method.

PubMed

Gutierre, R C; Vannucci Campos, D; Mortara, R A; Coppi, A A; Arida, R M

2017-04-01

Confocal laser-scanning microscopy is a useful tool for visualizing neurons and glia in transparent preparations of brain tissue from laboratory animals. Currently, imaging capillaries and venules in transparent brain tissues requires the use of fluorescent proteins. Here, we show that vessels can be imaged by confocal laser-scanning microscopy in transparent cortical, hippocampal and cerebellar preparations after clarification of China ink-injected specimens by the Spalteholz method. This method may be suitable for global, three-dimensional, quantitative analyses of vessels, including stereological estimations of total volume and length and of surface area of vessels, which constitute indirect approaches to investigate angiogenesis. © 2017 Anatomical Society.

A fast image simulation algorithm for scanning transmission electron microscopy.

PubMed

Ophus, Colin

2017-01-01

Image simulation for scanning transmission electron microscopy at atomic resolution for samples with realistic dimensions can require very large computation times using existing simulation algorithms. We present a new algorithm named PRISM that combines features of the two most commonly used algorithms, namely the Bloch wave and multislice methods. PRISM uses a Fourier interpolation factor f that has typical values of 4-20 for atomic resolution simulations. We show that in many cases PRISM can provide a speedup that scales with f 4 compared to multislice simulations, with a negligible loss of accuracy. We demonstrate the usefulness of this method with large-scale scanning transmission electron microscopy image simulations of a crystalline nanoparticle on an amorphous carbon substrate.

A fast image simulation algorithm for scanning transmission electron microscopy

DOE PAGES

Ophus, Colin

2017-05-10

Image simulation for scanning transmission electron microscopy at atomic resolution for samples with realistic dimensions can require very large computation times using existing simulation algorithms. Here, we present a new algorithm named PRISM that combines features of the two most commonly used algorithms, namely the Bloch wave and multislice methods. PRISM uses a Fourier interpolation factor f that has typical values of 4-20 for atomic resolution simulations. We show that in many cases PRISM can provide a speedup that scales with f 4 compared to multislice simulations, with a negligible loss of accuracy. We demonstrate the usefulness of this methodmore » with large-scale scanning transmission electron microscopy image simulations of a crystalline nanoparticle on an amorphous carbon substrate.« less

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

PubMed Central

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

2011-01-01

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

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy.

PubMed

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

2011-01-01

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

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

NASA Astrophysics Data System (ADS)

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

2011-01-01

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

Re-scan confocal microscopy: scanning twice for better resolution

PubMed Central

De Luca, Giulia M.R.; Breedijk, Ronald M.P.; Brandt, Rick A.J.; Zeelenberg, Christiaan H.C.; de Jong, Babette E.; Timmermans, Wendy; Azar, Leila Nahidi; Hoebe, Ron A.; Stallinga, Sjoerd; Manders, Erik M.M.

2013-01-01

We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required. PMID:24298422

Local carrier distribution imaging on few-layer MoS2 exfoliated on SiO2 by scanning nonlinear dielectric microscopy

NASA Astrophysics Data System (ADS)

Yamasue, Kohei; Cho, Yasuo

2018-06-01

We demonstrate that scanning nonlinear dielectric microscopy (SNDM) can be used for the nanoscale characterization of dominant carrier distribution on atomically thin MoS2 mechanically exfoliated on SiO2. For stable imaging without damaging microscopy tips and samples, SNDM was combined with peak-force tapping mode atomic force microscopy. The identification of dominant carriers and their spatial distribution becomes possible even for single and few-layer MoS2 on SiO2 using the proposed method allowing differential capacitance (dC/dV) imaging. We can expect that SNDM can also be applied to the evaluation of other two-dimensional semiconductors and devices.

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.

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

PubMed

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

2013-04-30

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

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy

PubMed Central

HÖHN, K.; FUCHS, J.; FRÖBER, A.; KIRMSE, R.; GLASS, B.; ANDERS‐ÖSSWEIN, M.; WALTHER, P.; KRÄUSSLICH, H.‐G.

2015-01-01

Summary In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV‐pulsed mature human dendritic cells. PMID:25786567

Scanning Ion Conductance Microscopy of Live Keratinocytes

NASA Astrophysics Data System (ADS)

Hegde, V.; Mason, A.; Saliev, T.; Smith, F. J. D.; McLean, W. H. I.; Campbell, P. A.

2012-07-01

Scanning ion conductance microscopy (SICM) is perhaps the least well known technique from the scanning probe microscopy (SPM) family of instruments. As with its more familiar counterpart, atomic force microscopy (AFM), the technique provides high-resolution topographic imaging, with the caveat that target structures must be immersed in a conducting solution so that a controllable ion current may be utilised as the basis for feedback. In operation, this non-contact characteristic of SICM makes it ideal for the study of delicate structures, such as live cells. Moreover, the intrinsic architecture of the instrument, incorporating as it does, a scanned micropipette, lends itself to combination approaches with complementary techniques such as patch-clamp electrophysiology: SICM therefore boasts the capability for both structural and functional imaging. For the present observations, an ICnano S system (Ionscope Ltd., Melbourn, UK) operating in 'hopping mode' was used, with the objective of assessing the instrument's utility for imaging live keratinocytes under physiological buffers. In scans employing cultured HaCaT cells (spontaneously immortalised, human keratinocytes), we compared the qualitative differences of live cells imaged with SICM and AFM, and also with their respective counterparts after chemical fixation in 4% paraformaldehyde. Characteristic surface microvilli were particularly prominent in live cell imaging by SICM. Moreover, time lapse SICM imaging on live cells revealed that changes in the pattern of microvilli could be tracked over time. By comparison, AFM imaging on live cells, even at very low contact forces (

Advantages of indium-tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells.

PubMed

Pluk, H; Stokes, D J; Lich, B; Wieringa, B; Fransen, J

2009-03-01

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium-tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.

Emulation and design of terahertz reflection-mode confocal scanning microscopy based on virtual pinhole

NASA Astrophysics Data System (ADS)

Yang, Yong-fa; Li, Qi

2014-12-01

In the practical application of terahertz reflection-mode confocal scanning microscopy, the size of detector pinhole is an important factor that determines the performance of spatial resolution characteristic of the microscopic system. However, the use of physical pinhole brings some inconvenience to the experiment and the adjustment error has a great influence on the experiment result. Through reasonably selecting the parameter of matrix detector virtual pinhole (VPH), it can efficiently approximate the physical pinhole. By using this approach, the difficulty of experimental calibration is reduced significantly. In this article, an imaging scheme of terahertz reflection-mode confocal scanning microscopy that is based on the matrix detector VPH is put forward. The influence of detector pinhole size on the axial resolution of confocal scanning microscopy is emulated and analyzed. Then, the parameter of VPH is emulated when the best axial imaging performance is reached.

Direct observation of iron-induced conformational changes of mitochondrial DNA by high-resolution field-emission in-lens scanning electron microscopy.

PubMed Central

Yaffee, M; Walter, P; Richter, C; Müller, M

1996-01-01

When respiring rat liver mitochondria are incubated in the presence of Fe(III) gluconate, their DNA (mtDNA) relaxes from the supercoiled to the open circular form dependent on the iron dose. Anaerobiosis or antioxidants fail to completely inhibit the unwinding. High-resolution field-emission in-lens scanning electron microscopy imaging, in concert with backscattered electron detection, pinpoints nanometer-range iron colloids bound to mtDNA isolated from iron-exposed mitochondria. High-resolution field-emission in-lens scanning electron microscopy with backscattered electron detection imaging permits simultaneous detailed visual analysis of DNA topology, iron dose-dependent mtDNA unwinding, and assessment of iron colloid formation on mtDNA strands. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8643576

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

PubMed

Höhn, K; Fuchs, J; Fröber, A; Kirmse, R; Glass, B; Anders-Össwein, M; Walther, P; Kräusslich, H-G; Dietrich, C

2015-08-01

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Functional Scanning Probe Imaging of Nanostructured Solar Energy Materials.

PubMed

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

2016-09-20

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

SPED light sheet microscopy: fast mapping of biological system structure and function

PubMed Central

Tomer, Raju; Lovett-Barron, Matthew; Kauvar, Isaac; Andalman, Aaron; Burns, Vanessa M.; Sankaran, Sethuraman; Grosenick, Logan; Broxton, Michael; Yang, Samuel; Deisseroth, Karl

2016-01-01

The goal of understanding living nervous systems has driven interest in high-speed and large field-of-view volumetric imaging at cellular resolution. Light-sheet microscopy approaches have emerged for cellular-resolution functional brain imaging in small organisms such as larval zebrafish, but remain fundamentally limited in speed. Here we have developed SPED light sheet microscopy, which combines large volumetric field-of-view via an extended depth of field with the optical sectioning of light sheet microscopy, thereby eliminating the need to physically scan detection objectives for volumetric imaging. SPED enables scanning of thousands of volumes-per-second, limited only by camera acquisition rate, through the harnessing of optical mechanisms that normally result in unwanted spherical aberrations. We demonstrate capabilities of SPED microscopy by performing fast sub-cellular resolution imaging of CLARITY mouse brains and cellular-resolution volumetric Ca2+ imaging of entire zebrafish nervous systems. Together, SPED light sheet methods enable high-speed cellular-resolution volumetric mapping of biological system structure and function. PMID:26687363

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

PubMed Central

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

2014-01-01

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

Chapter 14: Electron Microscopy on Thin Films for Solar Cells

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

Romero, Manuel; Abou-Ras, Daniel; Nichterwitz, Melanie

2016-07-22

This chapter overviews the various techniques applied in scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and highlights their possibilities and also limitations. It gives the various imaging and analysis techniques applied on a scanning electron microscope. The chapter shows that imaging is divided into that making use of secondary electrons (SEs) and of backscattered electrons (BSEs), resulting in different contrasts in the images and thus providing information on compositions, microstructures, and surface potentials. Whenever aiming for imaging and analyses at scales of down to the angstroms range, TEM and its related techniques are appropriate tools. In many cases,more » also SEM techniques provide the access to various material properties of the individual layers, not requiring specimen preparation as time consuming as TEM techniques. Finally, the chapter dedicates to cross-sectional specimen preparation for electron microscopy. The preparation decides indeed on the quality of imaging and analyses.« less

A hybrid scanning mode for fast scanning ion conductance microscopy (SICM) imaging

PubMed Central

Zhukov, Alex; Richards, Owen; Ostanin, Victor; Korchev, Yuri; Klenerman, David

2012-01-01

We have developed a new method of controlling the pipette for scanning ion conductance microscopy to obtain high-resolution images faster. The method keeps the pipette close to the surface during a single line scan but does not follow the exact surface topography, which is calculated by using the ion current. Using an FPGA platform we demonstrate this new method on model test samples and then on live cells. This method will be particularly useful to follow changes occurring on relatively flat regions of the cell surface at high spatial and temporal resolutions. PMID:22902298

Ultrafast photon counting applied to resonant scanning STED microscopy.

PubMed

Wu, Xundong; Toro, Ligia; Stefani, Enrico; Wu, Yong

2015-01-01

To take full advantage of fast resonant scanning in super-resolution stimulated emission depletion (STED) microscopy, we have developed an ultrafast photon counting system based on a multigiga sample per second analogue-to-digital conversion chip that delivers an unprecedented 450 MHz pixel clock (2.2 ns pixel dwell time in each scan). The system achieves a large field of view (∼50 × 50 μm) with fast scanning that reduces photobleaching, and advances the time-gated continuous wave STED technology to the usage of resonant scanning with hardware-based time-gating. The assembled system provides superb signal-to-noise ratio and highly linear quantification of light that result in superior image quality. Also, the system design allows great flexibility in processing photon signals to further improve the dynamic range. In conclusion, we have constructed a frontier photon counting image acquisition system with ultrafast readout rate, excellent counting linearity, and with the capacity of realizing resonant-scanning continuous wave STED microscopy with online time-gated detection. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

Microscanners for optical endomicroscopic applications

NASA Astrophysics Data System (ADS)

Hwang, Kyungmin; Seo, Yeong-Hyeon; Jeong, Ki-Hun

2017-12-01

MEMS laser scanning enables the miniaturization of endoscopic catheters for advanced endomicroscopy such as confocal microscopy, multiphoton microscopy, optical coherence tomography, and many other laser scanning microscopy. These advanced biomedical imaging modalities open a great potential for in vivo optical biopsy without surgical excision. They have huge capabilities for detecting on-demand early stage cancer with non-invasiveness. In this article, the scanning arrangement, trajectory, and actuation mechanism of endoscopic microscanners and their endomicroscopic applications will be overviewed.

Transmissive liquid-crystal device correcting primary coma aberration and astigmatism in laser scanning microscopy

NASA Astrophysics Data System (ADS)

Tanabe, Ayano; Hibi, Terumasa; Ipponjima, Sari; Matsumoto, Kenji; Yokoyama, Masafumi; Kurihara, Makoto; Hashimoto, Nobuyuki; Nemoto, Tomomi

2016-03-01

Laser scanning microscopy allows 3D cross-sectional imaging inside biospecimens. However, certain aberrations produced can degrade the quality of the resulting images. We previously reported a transmissive liquid-crystal device that could compensate for the predominant spherical aberrations during the observations, particularly in deep regions of the samples. The device, inserted between the objective lens and the microscope revolver, improved the image quality of fixed-mouse-brain slices that were observed using two-photon excitation laser scanning microscopy, which was originally degraded by spherical aberration. In this study, we developed a transmissive device that corrects primary coma aberration and astigmatism, motivated by the fact that these asymmetric aberrations can also often considerably deteriorate image quality, even near the sample surface. The device's performance was evaluated by observing fluorescent beads using single-photon excitation laser scanning microscopy. The fluorescence intensity in the image of the bead under a cover slip tilted in the y-direction was increased by 1.5 times after correction by the device. Furthermore, the y- and z-widths of the imaged bead were reduced to 66% and 65%, respectively. On the other hand, for the imaged bead sucked into a glass capillary in the longitudinal x-direction, correction with the device increased the fluorescence intensity by 2.2 times compared to that of the aberrated image. In addition, the x-, y-, and z-widths of the bead image were reduced to 75%, 53%, and 40%, respectively. Our device successfully corrected several asymmetric aberrations to improve the fluorescent signal and spatial resolution, and might be useful for observing various biospecimens.

Atomic force microscopy imaging of fragments from the Martian meteorite ALH84001

NASA Technical Reports Server (NTRS)

Steele, A.; Goddard, D.; Beech, I. B.; Tapper, R. C.; Stapleton, D.; Smith, J. R.

1998-01-01

A combination of scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) techniques, as well as atomic force microscopy (AFM) methods has been used to study fragments of the Martian meteorite ALH84001. Images of the same areas on the meteorite were obtained prior to and following gold/palladium coating by mapping the surface of the fragment using ESEM coupled with energy-dispersive X-ray analysis. Viewing of the fragments demonstrated the presence of structures, previously described as nanofossils by McKay et al. (Search for past life on Mars--possible relic biogenic activity in martian meteorite ALH84001. Science, 1996, pp. 924-930) of NASA who used SEM imaging of gold-coated meteorite samples. Careful imaging of the fragments revealed that the observed structures were not an artefact introduced by the coating procedure.

Scanning Probe Microscopy for Identifying the Component Materials of a Nanostripe Structure

NASA Astrophysics Data System (ADS)

Mizuno, Akira; Ando, Yasuhisa

2010-08-01

The authors prepared a nanostripe structure in which two types of metal are arranged alternately, and successfully identified the component materials using scanning probe microscopy (SPM) to measure the lateral force distribution image. The nanostripe structure was prepared using a new method developed by the authors and joint development members. The lateral force distribution image was measured in both friction force microscopy (FFM) and lateral modulation friction force microscopy (LM-FFM) modes. In FFM mode, the effect of slope angle appeared in the lateral force distribution image; therefore, no difference in the type of material was observed. On the other hand, in LM-FFM mode, the effect of surface curvature was observed in the lateral force distribution image. A higher friction force on chromium than on gold was identified, enabling material identification.

Correlative fluorescence and scanning transmission electron microscopy of quantum dot-labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Bandmann, Vera; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment. © 2014 Elsevier Inc. All rights reserved.

Carbon Nanotube Tip Probes: Stability and Lateral Resolution in Scanning Probe Microscopy and Application to Surface Science to Semiconductors

NASA Technical Reports Server (NTRS)

Nguyen, Cattien V.; Chao, Kuo-Jen; Stevens, Ramsey M. D.; Delzeit, Lance; Cassell, Alan; Han, Jie; Meyyappan, M.; Arnold, James (Technical Monitor)

2001-01-01

In this paper we present results on the stability and lateral resolution capability of carbon nanotube (CNT) scanning probes as applied to atomic force microscopy (AFM). Surface topography images of ultra-thin films (2-5 nm thickness) obtained with AFM are used to illustrate the lateral resolution capability of single-walled carbon nanotube probes. Images of metal films prepared by ion beam sputtering exhibit grain sizes ranging from greater than 10 nm to as small as approximately 2 nm for gold and iridium respectively. In addition, imaging stability and lifetime of multi-walled carbon nanotube scanning probes are studied on a relatively hard surface of silicon nitride (Si3N4). AFM images Of Si3N4 surface collected after more than 15 hrs of continuous scanning show no detectable degradation in lateral resolution. These results indicate the general feasibility of CNT tips and scanning probe microscopy for examining nanometer-scale surface features of deposited metals as well as non-conductive thin films. AFM coupled with CNT tips offers a simple and nondestructive technique for probing a variety of surfaces, and has immense potential as a surface characterization tool in integrated circuit manufacturing.

A fast MEMS scanning photoacoustic microscopy system and its application in glioma study

NASA Astrophysics Data System (ADS)

Bi, Renzhe; Balasundaram, Ghayathri; Jeon, Seungwan; Pu, Yang; Tay, Hui Chien; Kim, Chulhong; Olivo, Malini

2018-02-01

We present a water-proof Microelectromechanical systems (MEMS) based scanning optical resolution Photoacoustic Microscopy (OR-PAM) system and its application in glioma tumor mouse model study. The presented OR-PAM system has high optical resolution ( 3 μm) and high scanning speed (up to 50 kHz A-scan rate), which is ideal for cerebral vascular imaging. In this study, the mice with glioma tumor are treated with vascular disrupting agent (VDA). OR-PAM system is utilized to image the cerebral with the whole skull intact before and after the injection of VDA. By image registration, the response of every single blood vessel can be traced. This will provide us deeper understanding of the drug effect.

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

NASA Astrophysics Data System (ADS)

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

2018-06-01

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

Scanning tunneling spectroscopy under large current flow through the sample.

PubMed

Maldonado, A; Guillamón, I; Suderow, H; Vieira, S

2011-07-01

We describe a method to make scanning tunneling microscopy/spectroscopy imaging at very low temperatures while driving a constant electric current up to some tens of mA through the sample. It gives a new local probe, which we term current driven scanning tunneling microscopy/spectroscopy. We show spectroscopic and topographic measurements under the application of a current in superconducting Al and NbSe(2) at 100 mK. Perspective of applications of this local imaging method includes local vortex motion experiments, and Doppler shift local density of states studies.

An inexpensive approach for bright-field and dark-field imaging by scanning transmission electron microscopy in scanning electron microscopy.

PubMed

Patel, Binay; Watanabe, Masashi

2014-02-01

Scanning transmission electron microscopy in scanning electron microscopy (STEM-in-SEM) is a convenient technique for soft materials characterization. Various specimen-holder geometries and detector arrangements have been used for bright-field (BF) STEM-in-SEM imaging. In this study, to further the characterization potential of STEM-IN-SEM, a new specimen holder has been developed to facilitate direct detection of BF signals and indirect detection of dark-field (DF) signals without the need for substantial instrument modification. DF imaging is conducted with the use of a gold (Au)-coated copper (Cu) plate attached to the specimen holder which directs highly scattered transmitted electrons to an off-axis yttrium-aluminum-garnet (YAG) detector. A hole in the copper plate allows for BF imaging with a transmission electron (TE) detector. The inclusion of an Au-coated Cu plate enhanced DF signal intensity. Experiments validating the acquisition of true DF signals revealed that atomic number (Z) contrast may be achieved for materials with large lattice spacing. However, materials with small lattice spacing still exhibit diffraction contrast effects in this approach. The calculated theoretical fine probe size is 1.8 nm. At 30 kV, in this indirect approach, DF spatial resolution is limited to 3.2 nm as confirmed experimentally.

Quantitative single-molecule imaging by confocal laser scanning microscopy.

PubMed

Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

2008-11-25

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

Writing silica structures in liquid with scanning transmission electron microscopy.

PubMed

van de Put, Marcel W P; Carcouët, Camille C M C; Bomans, Paul H H; Friedrich, Heiner; de Jonge, Niels; Sommerdijk, Nico A J M

2015-02-04

Silica nanoparticles are imaged in solution with scanning transmission electron microscopy (STEM) using a liquid cell with silicon nitride (SiN) membrane windows. The STEM images reveal that silica structures are deposited in well-defined patches on the upper SiN membranes upon electron beam irradiation. The thickness of the deposits is linear with the applied electron dose. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrate that the deposited patches are a result of the merging of the original 20 nm-diameter nanoparticles, and that the related surface roughness depends on the electron dose rate used. Using this approach, sub-micrometer scale structures are written on the SiN in liquid by controlling the electron exposure as function of the lateral position. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sparse sampling and reconstruction for electron and scanning probe microscope imaging

DOEpatents

Anderson, Hyrum; Helms, Jovana; Wheeler, Jason W.; Larson, Kurt W.; Rohrer, Brandon R.

2015-07-28

Systems and methods for conducting electron or scanning probe microscopy are provided herein. In a general embodiment, the systems and methods for conducting electron or scanning probe microscopy with an undersampled data set include: driving an electron beam or probe to scan across a sample and visit a subset of pixel locations of the sample that are randomly or pseudo-randomly designated; determining actual pixel locations on the sample that are visited by the electron beam or probe; and processing data collected by detectors from the visits of the electron beam or probe at the actual pixel locations and recovering a reconstructed image of the sample.

Real time quantitative phase microscopy based on single-shot transport of intensity equation (ssTIE) method

NASA Astrophysics Data System (ADS)

Yu, Wei; Tian, Xiaolin; He, Xiaoliang; Song, Xiaojun; Xue, Liang; Liu, Cheng; Wang, Shouyu

2016-08-01

Microscopy based on transport of intensity equation provides quantitative phase distributions which opens another perspective for cellular observations. However, it requires multi-focal image capturing while mechanical and electrical scanning limits its real time capacity in sample detections. Here, in order to break through this restriction, real time quantitative phase microscopy based on single-shot transport of the intensity equation method is proposed. A programmed phase mask is designed to realize simultaneous multi-focal image recording without any scanning; thus, phase distributions can be quantitatively retrieved in real time. It is believed the proposed method can be potentially applied in various biological and medical applications, especially for live cell imaging.

Analysis of leaf surfaces using scanning ion conductance microscopy.

PubMed

Walker, Shaun C; Allen, Stephanie; Bell, Gordon; Roberts, Clive J

2015-05-01

Leaf surfaces are highly complex functional systems with well defined chemistry and structure dictating the barrier and transport properties of the leaf cuticle. It is a significant imaging challenge to analyse the very thin and often complex wax-like leaf cuticle morphology in their natural state. Scanning electron microscopy (SEM) and to a lesser extent Atomic force microscopy are techniques that have been used to study the leaf surface but their remains information that is difficult to obtain via these approaches. SEM is able to produce highly detailed and high-resolution images needed to study leaf structures at the submicron level. It typically operates in a vacuum or low pressure environment and as a consequence is generally unable to deal with the in situ analysis of dynamic surface events at submicron scales. Atomic force microscopy also possess the high-resolution imaging required and can follow dynamic events in ambient and liquid environments, but can over exaggerate small features and cannot image most leaf surfaces due to their inherent roughness at the micron scale. Scanning ion conductance microscopy (SICM), which operates in a liquid environment, provides a potential complementary analytical approach able to address these issues and which is yet to be explored for studying leaf surfaces. Here we illustrate the potential of SICM on various leaf surfaces and compare the data to SEM and atomic force microscopy images on the same samples. In achieving successful imaging we also show that SICM can be used to study the wetting of hydrophobic surfaces in situ. This has potentially wider implications than the study of leaves alone as surface wetting phenomena are important in a range of fundamental and applied studies. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Submicrometer fiber-optic chemical sensors: Measuring pH inside single cells

NASA Astrophysics Data System (ADS)

Kopelman, R.

Starting from scratch, we went in two and a half years to 0.04 micron optical microscopy resolution. We have demonstrated the application of near-field scanning optical microscopy to DNA samples and opened the new fields of near-field scanning spectroscopy and submicron opto-chemical sensors. All of these developments have been important steps towards in-situ DNA imaging and characterization on the nanoscale. Our first goal was to make NSOM (near-field scanning optical microscopy) a working enterprise, capable of 'zooming-in' towards a sample and imaging with a resolution exceeding that of traditional microscopy by a factor of ten. This has been achieved. Not only do we have a resolution of about 40 nm but we can image a 1 x 1 micron object in less than 10 seconds. Furthermore, the NSOM is a practical instrument. The tips survive for days or weeks of scanning and new methods of force feedback will soon protect the most fragile samples. Reproducible images of metal gratings, gold particles, dye balls (for calibration) and of several DNA samples have been made, proving the practicality of our approach. We also give highly resolved Force/NSOM images of human blood cells. Our second goal has been to form molecular optics (e.g., exciton donor) tips with a resolution of 2-10 nm for molecular excitation microscopy (MEM). We have produced such tips, and scanned with them, but only with a resolution comparable to that of our standard NSOM tips. However, we have demonstrated their potential for high resolution imaging capabilities: (1) An energy transfer (tip to sample) based feedback capability. (2) A Kasha (external heavy atom) effect based feedback. In addition, a novel and practical opto-chemical sensor that is a billion times smaller than the best ones available has been developed as well. Finally, we have also performed spatially resolved fluorescence spectroscopy.

A streaming multi-GPU implementation of image simulation algorithms for scanning transmission electron microscopy

DOE PAGES

Pryor, Alan; Ophus, Colin; Miao, Jianwei

2017-10-25

Simulation of atomic-resolution image formation in scanning transmission electron microscopy can require significant computation times using traditional methods. A recently developed method, termed plane-wave reciprocal-space interpolated scattering matrix (PRISM), demonstrates potential for significant acceleration of such simulations with negligible loss of accuracy. In this paper, we present a software package called Prismatic for parallelized simulation of image formation in scanning transmission electron microscopy (STEM) using both the PRISM and multislice methods. By distributing the workload between multiple CUDA-enabled GPUs and multicore processors, accelerations as high as 1000 × for PRISM and 15 × for multislice are achieved relative to traditionalmore » multislice implementations using a single 4-GPU machine. We demonstrate a potentially important application of Prismatic, using it to compute images for atomic electron tomography at sufficient speeds to include in the reconstruction pipeline. Prismatic is freely available both as an open-source CUDA/C++ package with a graphical user interface and as a Python package, PyPrismatic.« less

A streaming multi-GPU implementation of image simulation algorithms for scanning transmission electron microscopy.

PubMed

Pryor, Alan; Ophus, Colin; Miao, Jianwei

2017-01-01

Simulation of atomic-resolution image formation in scanning transmission electron microscopy can require significant computation times using traditional methods. A recently developed method, termed plane-wave reciprocal-space interpolated scattering matrix (PRISM), demonstrates potential for significant acceleration of such simulations with negligible loss of accuracy. Here, we present a software package called Prismatic for parallelized simulation of image formation in scanning transmission electron microscopy (STEM) using both the PRISM and multislice methods. By distributing the workload between multiple CUDA-enabled GPUs and multicore processors, accelerations as high as 1000 × for PRISM and 15 × for multislice are achieved relative to traditional multislice implementations using a single 4-GPU machine. We demonstrate a potentially important application of Prismatic , using it to compute images for atomic electron tomography at sufficient speeds to include in the reconstruction pipeline. Prismatic is freely available both as an open-source CUDA/C++ package with a graphical user interface and as a Python package, PyPrismatic .

A streaming multi-GPU implementation of image simulation algorithms for scanning transmission electron microscopy

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

Pryor, Alan; Ophus, Colin; Miao, Jianwei

Simulation of atomic-resolution image formation in scanning transmission electron microscopy can require significant computation times using traditional methods. A recently developed method, termed plane-wave reciprocal-space interpolated scattering matrix (PRISM), demonstrates potential for significant acceleration of such simulations with negligible loss of accuracy. In this paper, we present a software package called Prismatic for parallelized simulation of image formation in scanning transmission electron microscopy (STEM) using both the PRISM and multislice methods. By distributing the workload between multiple CUDA-enabled GPUs and multicore processors, accelerations as high as 1000 × for PRISM and 15 × for multislice are achieved relative to traditionalmore » multislice implementations using a single 4-GPU machine. We demonstrate a potentially important application of Prismatic, using it to compute images for atomic electron tomography at sufficient speeds to include in the reconstruction pipeline. Prismatic is freely available both as an open-source CUDA/C++ package with a graphical user interface and as a Python package, PyPrismatic.« less

The spatial coherence function in scanning transmission electron microscopy and spectroscopy.

PubMed

Nguyen, D T; Findlay, S D; Etheridge, J

2014-11-01

We investigate the implications of the form of the spatial coherence function, also referred to as the effective source distribution, for quantitative analysis in scanning transmission electron microscopy, and in particular for interpreting the spatial origin of imaging and spectroscopy signals. These questions are explored using three different source distribution models applied to a GaAs crystal case study. The shape of the effective source distribution was found to have a strong influence not only on the scanning transmission electron microscopy (STEM) image contrast, but also on the distribution of the scattered electron wavefield and hence on the spatial origin of the detected electron intensities. The implications this has for measuring structure, composition and bonding at atomic resolution via annular dark field, X-ray and electron energy loss STEM imaging are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

Towards ultrahigh resting-state functional connectivity in the mouse brain using photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Hariri, Ali; Bely, Nicholas; Chen, Chen; Nasiriavanaki, Mohammadreza

2016-03-01

The increasing use of mouse models for human brain disease studies, coupled with the fact that existing high-resolution functional imaging modalities cannot be easily applied to mice, presents an emerging need for a new functional imaging modality. Utilizing both mechanical and optical scanning in the photoacoustic microscopy, we can image spontaneous cerebral hemodynamic fluctuations and their associated functional connections in the mouse brain. The images is going to be acquired noninvasively with a fast frame rate, a large field of view, and a high spatial resolution. We developed an optical resolution photoacoustic microscopy (OR-PAM) with diode laser. Laser light was raster scanned due to XY-stage movement. Images from ultra-high OR-PAM can then be used to study brain disorders such as stroke, Alzheimer's, schizophrenia, multiple sclerosis, autism, and epilepsy.

Correlative Light and Scanning X-Ray Scattering Microscopy of Healthy and Pathologic Human Bone Sections

PubMed Central

Giannini, C.; Siliqi, D.; Bunk, O.; Beraudi, A.; Ladisa, M.; Altamura, D.; Stea, S.; Baruffaldi, F.

2012-01-01

Scanning small and wide angle X-ray scattering (scanning SWAXS) experiments were performed on healthy and pathologic human bone sections. Via crystallographic tools the data were transformed into quantitative images and as such compared with circularly polarized light (CPL) microscopy images. SWAXS and CPL images allowed extracting information of the mineral nanocrystalline phase embedded, with and without preferred orientation, in the collagen fibrils, mapping local changes at sub-osteon resolution. This favorable combination has been applied for the first time to biopsies of dwarfism syndrome and Paget's disease to shed light onto the cortical structure of natural bone in healthy and pathologic sections. PMID:22666538

Cycloid scanning for wide field optical coherence tomography endomicroscopy and angiography in vivo

PubMed Central

Liang, Kaicheng; Wang, Zhao; Ahsen, Osman O.; Lee, Hsiang-Chieh; Potsaid, Benjamin M.; Jayaraman, Vijaysekhar; Cable, Alex; Mashimo, Hiroshi; Li, Xingde; Fujimoto, James G.

2018-01-01

Devices that perform wide field-of-view (FOV) precision optical scanning are important for endoscopic assessment and diagnosis of luminal organ disease such as in gastroenterology. Optical scanning for in vivo endoscopic imaging has traditionally relied on one or more proximal mechanical actuators, limiting scan accuracy and imaging speed. There is a need for rapid and precise two-dimensional (2D) microscanning technologies to enable the translation of benchtop scanning microscopies to in vivo endoscopic imaging. We demonstrate a new cycloid scanner in a tethered capsule for ultrahigh speed, side-viewing optical coherence tomography (OCT) endomicroscopy in vivo. The cycloid capsule incorporates two scanners: a piezoelectrically actuated resonant fiber scanner to perform a precision, small FOV, fast scan and a micromotor scanner to perform a wide FOV, slow scan. Together these scanners distally scan the beam circumferentially in a 2D cycloid pattern, generating an unwrapped 1 mm × 38 mm strip FOV. Sequential strip volumes can be acquired with proximal pullback to image centimeter-long regions. Using ultrahigh speed 1.3 μm wavelength swept-source OCT at a 1.17 MHz axial scan rate, we imaged the human rectum at 3 volumes/s. Each OCT strip volume had 166 × 2322 axial scans with 8.5 μm axial and 30 μm transverse resolution. We further demonstrate OCT angiography at 0.5 volumes/s, producing volumetric images of vasculature. In addition to OCT applications, cycloid scanning promises to enable precision 2D optical scanning for other imaging modalities, including fluorescence confocal and nonlinear microscopy. PMID:29682598

Functional Scanning Probe Imaging of Nanostructured Solar Energy Materials

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

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

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

Functional Scanning Probe Imaging of Nanostructured Solar Energy Materials

DOE PAGES

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

2016-08-30

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

Correction of image drift and distortion in a scanning electron microscopy.

PubMed

Jin, P; Li, X

2015-12-01

Continuous research on small-scale mechanical structures and systems has attracted strong demand for ultrafine deformation and strain measurements. Conventional optical microscope cannot meet such requirements owing to its lower spatial resolution. Therefore, high-resolution scanning electron microscope has become the preferred system for high spatial resolution imaging and measurements. However, scanning electron microscope usually is contaminated by distortion and drift aberrations which cause serious errors to precise imaging and measurements of tiny structures. This paper develops a new method to correct drift and distortion aberrations of scanning electron microscope images, and evaluates the effect of correction by comparing corrected images with scanning electron microscope image of a standard sample. The drift correction is based on the interpolation scheme, where a series of images are captured at one location of the sample and perform image correlation between the first image and the consequent images to interpolate the drift-time relationship of scanning electron microscope images. The distortion correction employs the axial symmetry model of charged particle imaging theory to two images sharing with the same location of one object under different imaging fields of view. The difference apart from rigid displacement between the mentioned two images will give distortion parameters. Three-order precision is considered in the model and experiment shows that one pixel maximum correction is obtained for the employed high-resolution electron microscopic system. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

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.

Development of an add-on kit for scanning confocal microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Guo, Kaikai; Zheng, Guoan

2017-03-01

Scanning confocal microscopy is a standard choice for many fluorescence imaging applications in basic biomedical research. It is able to produce optically sectioned images and provide acquisition versatility to address many samples and application demands. However, scanning a focused point across the specimen limits the speed of image acquisition. As a result, scanning confocal microscope only works well with stationary samples. Researchers have performed parallel confocal scanning using digital-micromirror-device (DMD), which was used to project a scanning multi-point pattern across the sample. The DMD based parallel confocal systems increase the imaging speed while maintaining the optical sectioning ability. In this paper, we report the development of an add-on kit for high-speed and low-cost confocal microscopy. By adapting this add-on kit to an existing regular microscope, one can convert it into a confocal microscope without significant hardware modifications. Compared with current DMD-based implementations, the reported approach is able to recover multiple layers along the z axis simultaneously. It may find applications in wafer inspection and 3D metrology of semiconductor circuit. The dissemination of the proposed add-on kit under $1000 budget could also lead to new types of experimental designs for biological research labs, e.g., cytology analysis in cell culture experiments, genetic studies on multicellular organisms, pharmaceutical drug profiling, RNA interference studies, investigation of microbial communities in environmental systems, and etc.

Scanning two-photon microscopy with upconverting lanthanide nanoparticles via Richardson-Lucy deconvolution.

PubMed

Gainer, Christian F; Utzinger, Urs; Romanowski, Marek

2012-07-01

The use of upconverting lanthanide nanoparticles in fast-scanning microscopy is hindered by a long luminescence decay time, which greatly blurs images acquired in a nondescanned mode. We demonstrate herein an image processing method based on Richardson-Lucy deconvolution that mitigates the detrimental effects of their luminescence lifetime. This technique generates images with lateral resolution on par with the system's performance, ∼1.2  μm, while maintaining an axial resolution of 5 μm or better at a scan rate comparable with traditional two-photon microscopy. Remarkably, this can be accomplished with near infrared excitation power densities of 850 W/cm(2), several orders of magnitude below those used in two-photon imaging with molecular fluorophores. By way of illustration, we introduce the use of lipids to coat and functionalize these nanoparticles, rendering them water dispersible and readily conjugated to biologically relevant ligands, in this case epidermal growth factor receptor antibody. This deconvolution technique combined with the functionalized nanoparticles will enable three-dimensional functional tissue imaging at exceptionally low excitation power densities.

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

Sub-nanometre resolution imaging of polymer-fullerene photovoltaic blends using energy-filtered scanning electron microscopy.

PubMed

Masters, Robert C; Pearson, Andrew J; Glen, Tom S; Sasam, Fabian-Cyril; Li, Letian; Dapor, Maurizio; Donald, Athene M; Lidzey, David G; Rodenburg, Cornelia

2015-04-24

The resolution capability of the scanning electron microscope has increased immensely in recent years, and is now within the sub-nanometre range, at least for inorganic materials. An equivalent advance has not yet been achieved for imaging the morphologies of nanostructured organic materials, such as organic photovoltaic blends. Here we show that energy-selective secondary electron detection can be used to obtain high-contrast, material-specific images of an organic photovoltaic blend. We also find that we can differentiate mixed phases from pure material phases in our data. The lateral resolution demonstrated is twice that previously reported from secondary electron imaging. Our results suggest that our energy-filtered scanning electron microscopy approach will be able to make major inroads into the understanding of complex, nano-structured organic materials.

Confocal microscopy imaging of solid tissue

EPA Science Inventory

Confocal laser scanning microscopy (CLSM) is a technique that is capable of generating serial sections of whole-mount tissue and then reassembling the computer acquired images as a virtual 3-dimensional structure. In many ways CLSM offers an alternative to traditional sectioning ...

Multi-scale Imaging of Cellular and Sub-cellular Structures using Scanning Probe Recognition Microscopy.

NASA Astrophysics Data System (ADS)

Chen, Q.; Rice, A. F.

2005-03-01

Scanning Probe Recognition Microscopy is a new scanning probe capability under development within our group to reliably return to and directly interact with a specific nanobiological feature of interest. In previous work, we have successfully recognized and classified tubular versus globular biological objects from experimental atomic force microscope images using a method based on normalized central moments [ref. 1]. In this paper we extend this work to include recognition schemes appropriate for cellular and sub-cellular structures. Globular cells containing tubular actin filaments are under investigation. Thus there are differences in external/internal shapes and scales. Continuous Wavelet Transform with a differential Gaussian mother wavelet is employed for multi- scale analysis. [ref. 1] Q. Chen, V. Ayres and L. Udpa, ``Biological Investigation Using Scanning Probe Recognition Microscopy,'' Proceedings 3rd IEEE Conference on Nanotechnology, vol. 2, p 863-865 (2003).

USE OF MULTIPHOTON LASER SCANNING MICROSCOPY TO IMAGE BENZO[A]PYRENE AND METABOLITES IN FISH EGGS

EPA Science Inventory

Multiphoton laser scanning microscopy (MPLSM) is a promising tool to study the tissue distribution of environmental chemical contaminants during fish early life stages. One such chemical for which this is possible is benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon that a...

Multi-modal Registration for Correlative Microscopy using Image Analogies

PubMed Central

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

2014-01-01

Correlative microscopy is a methodology combining the functionality of light microscopy with the high resolution of electron microscopy and other microscopy technologies for the same biological specimen. In this paper, we propose an image registration method for correlative microscopy, which is challenging due to the distinct appearance of biological structures when imaged with different modalities. Our method is based on image analogies and allows to transform images of a given modality into the appearance-space of another modality. Hence, the registration between two different types of microscopy images can be transformed to a mono-modality image registration. We use a sparse representation model to obtain image analogies. The method makes use of corresponding image training patches of two different imaging modalities to learn a dictionary capturing appearance relations. We test our approach on backscattered electron (BSE) scanning electron microscopy (SEM)/confocal and transmission electron microscopy (TEM)/confocal images. We perform rigid, affine, and deformable registration via B-splines and show improvements over direct registration using both mutual information and sum of squared differences similarity measures to account for differences in image appearance. PMID:24387943

Applications of microscopy to genetic therapy of cystic fibrosis and other human diseases.

PubMed

Moninger, Thomas O; Nessler, Randy A; Moore, Kenneth C

2006-01-01

Gene therapy has become an extremely important and active field of biomedical research. Microscopy is an integral component of this effort. This chapter presents an overview of imaging techniques used in our facility in support of cystic fibrosis gene therapy research. Instrumentation used in these studies includes light and confocal microscopy, transmission electron microscopy, and scanning electron microscopy. Techniques outlined include negative staining, cryo-electron microscopy, three-dimentional reconstruction, enzyme cytochemistry, immunocytochemistry, and fluorescence imaging.

X-ray microscopy as an approach to increasing accuracy and efficiency of serial block-face imaging for correlated light and electron microscopy of biological specimens.

PubMed

Bushong, Eric A; Johnson, Donald D; Kim, Keun-Young; Terada, Masako; Hatori, Megumi; Peltier, Steven T; Panda, Satchidananda; Merkle, Arno; Ellisman, Mark H

2015-02-01

The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.

X-ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-face Imaging for Correlated Light and Electron Microscopy of Biological Specimens

PubMed Central

Bushong, Eric A.; Johnson, Donald D.; Kim, Keun-Young; Terada, Masako; Hatori, Megumi; Peltier, Steven T.; Panda, Satchidananda; Merkle, Arno; Ellisman, Mark H.

2015-01-01

The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging. PMID:25392009

Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging.

PubMed

Thériault, Gabrielle; Cottet, Martin; Castonguay, Annie; McCarthy, Nathalie; De Koninck, Yves

2014-01-01

Two-photon microscopy has revolutionized functional cellular imaging in tissue, but although the highly confined depth of field (DOF) of standard set-ups yields great optical sectioning, it also limits imaging speed in volume samples and ease of use. For this reason, we recently presented a simple and retrofittable modification to the two-photon laser-scanning microscope which extends the DOF through the use of an axicon (conical lens). Here we demonstrate three significant benefits of this technique using biological samples commonly employed in the field of neuroscience. First, we use a sample of neurons grown in culture and move it along the z-axis, showing that a more stable focus is achieved without compromise on transverse resolution. Second, we monitor 3D population dynamics in an acute slice of live mouse cortex, demonstrating that faster volumetric scans can be conducted. Third, we acquire a stereoscopic image of neurons and their dendrites in a fixed sample of mouse cortex, using only two scans instead of the complete stack and calculations required by standard systems. Taken together, these advantages, combined with the ease of integration into pre-existing systems, make the extended depth-of-field imaging based on Bessel beams a strong asset for the field of microscopy and life sciences in general.

Fabrication of [001]-oriented tungsten tips for high resolution scanning tunneling microscopy

PubMed Central

Chaika, A. N.; Orlova, N. N.; Semenov, V. N.; Postnova, E. Yu.; Krasnikov, S. A.; Lazarev, M. G.; Chekmazov, S. V.; Aristov, V. Yu.; Glebovsky, V. G.; Bozhko, S. I.; Shvets, I. V.

2014-01-01

The structure of the [001]-oriented single crystalline tungsten probes sharpened in ultra-high vacuum using electron beam heating and ion sputtering has been studied using scanning and transmission electron microscopy. The electron microscopy data prove reproducible fabrication of the single-apex tips with nanoscale pyramids grained by the {011} planes at the apexes. These sharp, [001]-oriented tungsten tips have been successfully utilized in high resolution scanning tunneling microscopy imaging of HOPG(0001), SiC(001) and graphene/SiC(001) surfaces. The electron microscopy characterization performed before and after the high resolution STM experiments provides direct correlation between the tip structure and picoscale spatial resolution achieved in the experiments. PMID:24434734

Optimizing pulse compressibility in completely all-fibered Ytterbium chirped pulse amplifiers for in vivo two photon laser scanning microscopy

PubMed Central

Fernández, A.; Grüner-Nielsen, L.; Andreana, M.; Stadler, M.; Kirchberger, S.; Sturtzel, C.; Distel, M.; Zhu, L.; Kautek, W.; Leitgeb, R.; Baltuska, A.; Jespersen, K.; Verhoef, A.

2017-01-01

A simple and completely all-fiber Yb chirped pulse amplifier that uses a dispersion matched fiber stretcher and a spliced-on hollow core photonic bandgap fiber compressor is applied in nonlinear optical microscopy. This stretching-compression approach improves compressibility and helps to maximize the fluorescence signal in two-photon laser scanning microscopy as compared with approaches that use standard single mode fibers as stretcher. We also show that in femtosecond all-fiber systems, compensation of higher order dispersion terms is relevant even for pulses with relatively narrow bandwidths for applications relying on nonlinear optical effects. The completely all-fiber system was applied to image green fluorescent beads, a stained lily-of-the-valley root and rat-tail tendon. We also demonstrated in vivo imaging in zebrafish larvae, where we simultaneously measure second harmonic and fluorescence from two-photon excited red-fluorescent protein. Since the pulses are compressed in a fiber, this source is especially suited for upgrading existing laser scanning (confocal) microscopes with multiphoton imaging capabilities in space restricted settings or for incorporation in endoscope-based microscopy. PMID:28856032

Optimizing pulse compressibility in completely all-fibered Ytterbium chirped pulse amplifiers for in vivo two photon laser scanning microscopy.

PubMed

Fernández, A; Grüner-Nielsen, L; Andreana, M; Stadler, M; Kirchberger, S; Sturtzel, C; Distel, M; Zhu, L; Kautek, W; Leitgeb, R; Baltuska, A; Jespersen, K; Verhoef, A

2017-08-01

A simple and completely all-fiber Yb chirped pulse amplifier that uses a dispersion matched fiber stretcher and a spliced-on hollow core photonic bandgap fiber compressor is applied in nonlinear optical microscopy. This stretching-compression approach improves compressibility and helps to maximize the fluorescence signal in two-photon laser scanning microscopy as compared with approaches that use standard single mode fibers as stretcher. We also show that in femtosecond all-fiber systems, compensation of higher order dispersion terms is relevant even for pulses with relatively narrow bandwidths for applications relying on nonlinear optical effects. The completely all-fiber system was applied to image green fluorescent beads, a stained lily-of-the-valley root and rat-tail tendon. We also demonstrated in vivo imaging in zebrafish larvae, where we simultaneously measure second harmonic and fluorescence from two-photon excited red-fluorescent protein. Since the pulses are compressed in a fiber, this source is especially suited for upgrading existing laser scanning (confocal) microscopes with multiphoton imaging capabilities in space restricted settings or for incorporation in endoscope-based microscopy.

Correlative 3D imaging of Whole Mammalian Cells with Light and Electron Microscopy

PubMed Central

Murphy, Gavin E.; Narayan, Kedar; Lowekamp, Bradley C.; Hartnell, Lisa M.; Heymann, Jurgen A. W.; Fu, Jing; Subramaniam, Sriram

2011-01-01

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA–SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ~10 to 20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA–SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues. PMID:21907806

Novel medical image enhancement algorithms

NASA Astrophysics Data System (ADS)

Agaian, Sos; McClendon, Stephen A.

2010-01-01

In this paper, we present two novel medical image enhancement algorithms. The first, a global image enhancement algorithm, utilizes an alpha-trimmed mean filter as its backbone to sharpen images. The second algorithm uses a cascaded unsharp masking technique to separate the high frequency components of an image in order for them to be enhanced using a modified adaptive contrast enhancement algorithm. Experimental results from enhancing electron microscopy, radiological, CT scan and MRI scan images, using the MATLAB environment, are then compared to the original images as well as other enhancement methods, such as histogram equalization and two forms of adaptive contrast enhancement. An image processing scheme for electron microscopy images of Purkinje cells will also be implemented and utilized as a comparison tool to evaluate the performance of our algorithm.

Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy.

PubMed

Schulz, Olaf; Pieper, Christoph; Clever, Michaela; Pfaff, Janine; Ruhlandt, Aike; Kehlenbach, Ralph H; Wouters, Fred S; Großhans, Jörg; Bunt, Gertrude; Enderlein, Jörg

2013-12-24

We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.

Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy

PubMed Central

Schulz, Olaf; Pieper, Christoph; Clever, Michaela; Pfaff, Janine; Ruhlandt, Aike; Kehlenbach, Ralph H.; Wouters, Fred S.; Großhans, Jörg; Bunt, Gertrude; Enderlein, Jörg

2013-01-01

We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions. PMID:24324140

Two-photon speckle illumination for super-resolution microscopy.

PubMed

Negash, Awoke; Labouesse, Simon; Chaumet, Patrick C; Belkebir, Kamal; Giovannini, Hugues; Allain, Marc; Idier, Jérôme; Sentenac, Anne

2018-06-01

We present a numerical study of a microscopy setup in which the sample is illuminated with uncontrolled speckle patterns and the two-photon excitation fluorescence is collected on a camera. We show that, using a simple deconvolution algorithm for processing the speckle low-resolution images, this wide-field imaging technique exhibits resolution significantly better than that of two-photon excitation scanning microscopy or one-photon excitation bright-field microscopy.

Atomic resolution elemental mapping using energy-filtered imaging scanning transmission electron microscopy with chromatic aberration correction.

PubMed

Krause, F F; Rosenauer, A; Barthel, J; Mayer, J; Urban, K; Dunin-Borkowski, R E; Brown, H G; Forbes, B D; Allen, L J

2017-10-01

This paper addresses a novel approach to atomic resolution elemental mapping, demonstrating a method that produces elemental maps with a similar resolution to the established method of electron energy-loss spectroscopy in scanning transmission electron microscopy. Dubbed energy-filtered imaging scanning transmission electron microscopy (EFISTEM) this mode of imaging is, by the quantum mechanical principle of reciprocity, equivalent to tilting the probe in energy-filtered transmission electron microscopy (EFTEM) through a cone and incoherently averaging the results. In this paper we present a proof-of-principle EFISTEM experimental study on strontium titanate. The present approach, made possible by chromatic aberration correction, has the advantage that it provides elemental maps which are immune to spatial incoherence in the electron source, coherent aberrations in the probe-forming lens and probe jitter. The veracity of the experiment is supported by quantum mechanical image simulations, which provide an insight into the image-forming process. Elemental maps obtained in EFTEM suffer from the effect known as preservation of elastic contrast, which, for example, can lead to a given atomic species appearing to be in atomic columns where it is not to be found. EFISTEM very substantially reduces the preservation of elastic contrast and yields images which show stability of contrast with changing thickness. The experimental application is demonstrated in a proof-of-principle study on strontium titanate. Copyright © 2017 Elsevier B.V. All rights reserved.

Understanding Imaging and Metrology with the Helium Ion Microscope

NASA Astrophysics Data System (ADS)

Postek, Michael T.; Vladár, András E.; Ming, Bin

2009-09-01

One barrier to innovation confronting all phases of nanotechnology is the lack of accurate metrology for the characterization of nanomaterials. Ultra-high resolution microscopy is a key technology needed to achieve this goal. But, current microscope technology is being pushed to its limits. The scanning and transmission electron microscopes have incrementally improved in performance and other scanned probe technologies such as atomic force microscopy, scanning tunneling microscopy and focused ion beam microscopes have all been applied to nanotechnology with various levels of success. A relatively new tool for nanotechnology is the scanning helium ion microscope (HIM). The HIM is a new complementary imaging and metrology technology for nanotechnology which may be able to push the current resolution barrier lower. But, successful imaging and metrology with this instrument entails new ion beam/specimen interaction physics which must be fully understood. As a new methodology, HIM is beginning to show promise and the abundance of potentially advantageous applications for nanotechnology have yet to be fully exploited. This presentation will discuss some of the progress made at NIST in understanding the science behind this new technique.

Phase contrast scanning transmission electron microscopy imaging of light and heavy atoms at the limit of contrast and resolution.

PubMed

Yücelen, Emrah; Lazić, Ivan; Bosch, Eric G T

2018-02-08

Using state of the art scanning transmission electron microscopy (STEM) it is nowadays possible to directly image single atomic columns at sub-Å resolution. In standard (high angle) annular dark field STEM ((HA)ADF-STEM), however, light elements are usually invisible when imaged together with heavier elements in one image. Here we demonstrate the capability of the recently introduced Integrated Differential Phase Contrast STEM (iDPC-STEM) technique to image both light and heavy atoms in a thin sample at sub-Å resolution. We use the technique to resolve both the Gallium and Nitrogen dumbbells in a GaN crystal in [[Formula: see text

Investigation of Electron Transport Across Vertically Grown CNTs Using Combination of Proximity Field Emission Microscopy and Scanning Probe Image Processing Techniques

NASA Astrophysics Data System (ADS)

Kolekar, Sadhu; Patole, Shashikant P.; Yoo, Ji-Beom; Dharmadhikari, Chandrakant V.

2018-03-01

Field emission from nanostructured films is known to be dominated by only small number of localized spots which varies with the voltage, electric field and heat treatment. It is important to develop processing methods which will produce stable and uniform emitting sites. In this paper we report a novel approach which involves analysis of Proximity Field Emission Microscopic (PFEM) images using Scanning Probe Image Processing technique. Vertically aligned carbon nanotube emitters have been deposited on tungsten foil by water assisted chemical vapor deposition. Prior to the field electron emission studies, these films were characterized by scanning electron microscopy, transmission electron microscopy, and Atomic Force Microscopy (AFM). AFM images of the samples show bristle like structure, the size of bristle varying from 80 to 300 nm. The topography images were found to exhibit strong correlation with current images. Current-Voltage (I-V) measurements both from Scanning Tunneling Microscopy and Conducting-AFM mode suggest that electron transport mechanism in imaging vertically grown CNTs is ballistic rather than usual tunneling or field emission with a junction resistance of 10 kΩ. It was found that I-V curves for field emission mode in PFEM geometry vary initially with number of I-V cycles until reproducible I-V curves are obtained. Even for reasonably stable I-V behavior the number of spots was found to increase with the voltage leading to a modified Fowler-Nordheim (F-N) behavior. A plot of ln(I/V3) versus 1/V was found to be linear. Current versus time data exhibit large fluctuation with the power spectral density obeying 1/f2 law. It is suggested that an analogue of F-N equation of the form ln(I/Vα) versus 1/V may be used for the analysis of field emission data, where α may depend on nanostructure configuration and can be determined from the dependence of emitting spots on the voltage.

Microscopy using source and detector arrays

NASA Astrophysics Data System (ADS)

Sheppard, Colin J. R.; Castello, Marco; Vicidomini, Giuseppe; Duocastella, Martí; Diaspro, Alberto

2016-03-01

There are basically two types of microscope, which we call conventional and scanning. The former type is a full-field imaging system. In the latter type, the object is illuminated with a probe beam, and a signal detected. We can generalize the probe to a patterned illumination. Similarly we can generalize the detection to a patterned detection. Combining these we get a range of different modalities: confocal microscopy, structured illumination (with full-field imaging), spinning disk (with multiple illumination points), and so on. The combination allows the spatial frequency bandwidth of the system to be doubled. In general we can record a four dimensional (4D) image of a 2D object (or a 6D image from a 3D object, using an acoustic tuneable lens). The optimum way to directly reconstruct the resulting image is by image scanning microscopy (ISM). But the 4D image is highly redundant, so deconvolution-based approaches are also relevant. ISM can be performed in fluorescence, bright field or interference microscopy. Several different implementations have been described, with associated advantages and disadvantages. In two-photon microscopy, the illumination and detection point spread functions are very different. This is also the case when using pupil filters or when there is a large Stokes shift.

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy

PubMed Central

Lerner, Thomas R.; Burden, Jemima J.; Nkwe, David O.; Pelchen-Matthews, Annegret; Domart, Marie-Charlotte; Durgan, Joanne; Weston, Anne; Jones, Martin L.; Peddie, Christopher J.; Carzaniga, Raffaella; Florey, Oliver; Marsh, Mark; Gutierrez, Maximiliano G.

2017-01-01

ABSTRACT The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research. PMID:27445312

The Scanning Electron Microscope and the Archaeologist

ERIC Educational Resources Information Center

Ponting, Matthew

2004-01-01

Images from scanning electron microscopy are now quite common and they can be of great value in archaeology. Techniques such as secondary electron imaging, backscattered electron imaging and energy-dispersive x-ray analysis can reveal information such as the presence of weevils in grain in Roman Britain, the composition of Roman coins and the…

Preparation of isolated biomolecules for SFM observations: T4 bacteriophage as a test sample.

PubMed Central

Droz, E; Taborelli, M; Wells, T N; Descouts, P

1993-01-01

The T4 bacteriophage has been used to investigate protocols for the preparation of samples for scanning force microscopy in air, in order to obtaining reproducible images. The resolution of images and the distribution of bacteriophages on the substrate depends on the buffer type, its concentration, the surface treatment of substrate, and the method of deposition. The best imaging conditions for the phages require dilution in a volatile buffer at low ionic strength and adsorption onto hydrophilic surfaces. When imaging with the scanning force microscopy the quality of the images is influenced by the vertical and lateral forces applied on the sample and by the tip geometry. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 6 PMID:8241398

Analysis of Multilayer Devices for Superconducting Electronics by High-Resolution Scanning Transmission Electron Microscopy and Energy Dispersive Spectroscopy

DOE PAGES

Missert, Nancy; Kotula, Paul G.; Rye, Michael; ...

2017-02-15

We used a focused ion beam to obtain cross-sectional specimens from both magnetic multilayer and Nb/Al-AlOx/Nb Josephson junction devices for characterization by scanning transmission electron microscopy (STEM) and energy dispersive X-ray spectroscopy (EDX). An automated multivariate statistical analysis of the EDX spectral images produced chemically unique component images of individual layers within the multilayer structures. STEM imaging elucidated distinct variations in film morphology, interface quality, and/or etch artifacts that could be correlated to magnetic and/or electrical properties measured on the same devices.

A new scanning electron microscopy approach to image aerogels at the nanoscale

NASA Astrophysics Data System (ADS)

Solá, F.; Hurwitz, F.; Yang, J.

2011-04-01

A new scanning electron microscopy (SEM) technique to image poor electrically conductive aerogels is presented. The process can be performed by non-expert SEM users. We showed that negative charging effects on aerogels can be minimized significantly by inserting dry nitrogen gas close to the region of interest. The process involves the local recombination of accumulated negative charges with positive ions generated from ionization processes. This new technique made possible the acquisition of images of aerogels with pores down to approximately 3 nm in diameter using a positively biased Everhart-Thornley (ET) detector.

High-resolution low-dose scanning transmission electron microscopy.

PubMed

Buban, James P; Ramasse, Quentin; Gipson, Bryant; Browning, Nigel D; Stahlberg, Henning

2010-01-01

During the past two decades instrumentation in scanning transmission electron microscopy (STEM) has pushed toward higher intensity electron probes to increase the signal-to-noise ratio of recorded images. While this is suitable for robust specimens, biological specimens require a much reduced electron dose for high-resolution imaging. We describe here protocols for low-dose STEM image recording with a conventional field-emission gun STEM, while maintaining the high-resolution capability of the instrument. Our findings show that a combination of reduced pixel dwell time and reduced gun current can achieve radiation doses comparable to low-dose TEM.

Effects of pupil filter patterns in line-scan focal modulation microscopy

NASA Astrophysics Data System (ADS)

Shen, Shuhao; Pant, Shilpa; Chen, Rui; Chen, Nanguang

2018-03-01

Line-scan focal modulation microscopy (LSFMM) is an emerging imaging technique that affords high imaging speed and good optical sectioning at the same time. We present a systematic investigation into optimal design of the pupil filter for LSFMM in an attempt to achieve the best performance in terms of spatial resolutions, optical sectioning, and modulation depth. Scalar diffraction theory was used to compute light propagation and distribution in the system and theoretical predictions on system performance, which were then compared with experimental results.

Special raster scanning for reduction of charging effects in scanning electron microscopy.

PubMed

Suzuki, Kazuhiko; Oho, Eisaku

2014-01-01

A special raster scanning (SRS) method for reduction of charging effects is developed for the field of SEM. Both a conventional fast scan (horizontal direction) and an unusual scan (vertical direction) are adopted for acquiring raw data consisting of many sub-images. These data are converted to a proper SEM image using digital image processing techniques. About sharpness of the image and reduction of charging effects, the SRS is compared with the conventional fast scan (with frame-averaging) and the conventional slow scan. Experimental results show the effectiveness of SRS images. By a successful combination of the proposed scanning method and low accelerating voltage (LV)-SEMs, it is expected that higher-quality SEM images can be more easily acquired by the considerable reduction of charging effects, while maintaining the resolution. © 2013 Wiley Periodicals, Inc.

Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film.

PubMed

Nishiyama, Hidetoshi; Suga, Mitsuo; Ogura, Toshihiko; Maruyama, Yuusuke; Koizumi, Mitsuru; Mio, Kazuhiro; Kitamura, Shinichi; Sato, Chikara

2010-03-01

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry. (c) 2010 Elsevier Inc. All rights reserved.

Alpha particle spectroscopy using FNTD and SIM super-resolution microscopy.

PubMed

Kouwenberg, J J M; Kremers, G J; Slotman, J A; Wolterbeek, H T; Houtsmuller, A B; Denkova, A G; Bos, A J J

2018-06-01

Structured illumination microscopy (SIM) for the imaging of alpha particle tracks in fluorescent nuclear track detectors (FNTD) was evaluated and compared to confocal laser scanning microscopy (CLSM). FNTDs were irradiated with an external alpha source and imaged using both methodologies. SIM imaging resulted in improved resolution, without increase in scan time. Alpha particle energy estimation based on the track length, direction and intensity produced results in good agreement with the expected alpha particle energy distribution. A pronounced difference was seen in the spatial scattering of alpha particles in the detectors, where SIM showed an almost 50% reduction compared to CLSM. The improved resolution of SIM allows for more detailed studies of the tracks induced by ionising particles. The combination of SIM and FNTDs for alpha radiation paves the way for affordable and fast alpha spectroscopy and dosimetry. © 2018 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Scanning electron microscopy of cells and tissues under fully hydrated conditions

PubMed Central

Thiberge, Stephan; Nechushtan, Amotz; Sprinzak, David; Gileadi, Opher; Behar, Vered; Zik, Ory; Chowers, Yehuda; Michaeli, Shulamit; Schlessinger, Joseph; Moses, Elisha

2004-01-01

A capability for scanning electron microscopy of wet biological specimens is presented. A membrane that is transparent to electrons protects the fully hydrated sample from the vacuum. The result is a hybrid technique combining the ease of use and ability to see into cells of optical microscopy with the higher resolution of electron microscopy. The resolution of low-contrast materials is ≈100 nm, whereas in high-contrast materials the resolution can reach 10 nm. Standard immunogold techniques and heavy-metal stains can be applied and viewed in the fluid to improve the contrast. Images present a striking combination of whole-cell morphology with a wealth of internal details. A possibility for direct inspection of tissue slices transpires, imaging only the external layer of cells. Simultaneous imaging with photons excited by the electrons incorporates data on material distribution, indicating a potential for multilabeling and specific scintillating markers. PMID:14988502

Sub-nanometre resolution imaging of polymer–fullerene photovoltaic blends using energy-filtered scanning electron microscopy

PubMed Central

Masters, Robert C.; Pearson, Andrew J.; Glen, Tom S.; Sasam, Fabian-Cyril; Li, Letian; Dapor, Maurizio; Donald, Athene M.; Lidzey, David G.; Rodenburg, Cornelia

2015-01-01

The resolution capability of the scanning electron microscope has increased immensely in recent years, and is now within the sub-nanometre range, at least for inorganic materials. An equivalent advance has not yet been achieved for imaging the morphologies of nanostructured organic materials, such as organic photovoltaic blends. Here we show that energy-selective secondary electron detection can be used to obtain high-contrast, material-specific images of an organic photovoltaic blend. We also find that we can differentiate mixed phases from pure material phases in our data. The lateral resolution demonstrated is twice that previously reported from secondary electron imaging. Our results suggest that our energy-filtered scanning electron microscopy approach will be able to make major inroads into the understanding of complex, nano-structured organic materials. PMID:25906738

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2015-09-11

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results.

Video-rate in vivo fluorescence imaging with a line-scanned dual-axis confocal microscope.

PubMed

Chen, Ye; Wang, Danni; Khan, Altaz; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T C

2015-10-01

Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

Video-rate in vivo fluorescence imaging with a line-scanned dual-axis confocal microscope

NASA Astrophysics Data System (ADS)

Chen, Ye; Wang, Danni; Khan, Altaz; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T. C.

2015-10-01

Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

Localized electronic structures of graphene oxide studied using scanning tunneling microscopy and spectroscopy.

PubMed

Katano, Satoshi; Wei, Tao; Sasajima, Takumi; Kasama, Ryuhei; Uehara, Yoichi

2018-06-21

We have used scanning tunneling microscopy (STM) to elucidate the nanoscale electronic structures of graphene oxide (GO). The unreduced GO layer was imaged using STM without reduction processes when deposited on a Au(111) surface covered with an octanethiolate self-assembled monolayer (C8S-SAM). The STM image of the GO sheet exhibits a grainy structure having a thickness of about 1 nm, which is in good agreement with the previous results obtained using atomic force microscopy (AFM). We found that the C8S-SAM suppresses the adsorption of water remaining on the substrate, which would be important to accomplish the nanoscale imaging of the unreduced GO by STM. Furthermore, we successfully detected the π and π* states localized in the GO sheet using scanning tunneling spectroscopy (STS). The π-π* gap energy and the gap center are not uniform within the GO sheet, indicating the existence of various sizes of the sp2 domain and evidence for the local electronic doping by the substituents.

Nanomorphology of P3HT:PCBM-based absorber layers of organic solar cells after different processing conditions analyzed by low-energy scanning transmission electron microscopy.

PubMed

Pfaff, Marina; Klein, Michael F G; Müller, Erich; Müller, Philipp; Colsmann, Alexander; Lemmer, Uli; Gerthsen, Dagmar

2012-12-01

In this study the nanomorphology of P3HT:PC61BM absorber layers of organic solar cells was studied as a function of the processing parameters and for P3HT with different molecular weight. For this purpose we apply scanning transmission electron microscopy (STEM) at low electron energies in a scanning electron microscope. This method exhibits sensitive material contrast in the high-angle annular dark-field (HAADF) mode, which is well suited to distinguish materials with similar densities and mean atomic numbers. The images taken with low-energy HAADF STEM are compared with conventional transmission electron microscopy and atomic force microscopy images to illustrate the capabilities of the different techniques. For the interpretation of the low-energy HAADF STEM images, a semiempirical equation is used to calculate the image intensities. The experiments show that the nanomorphology of the P3HT:PC61BM blends depends strongly on the molecular weight of the P3HT. Low-molecular-weight P3HT forms rod-like domains during annealing. In contrast, only small globular features are visible in samples containing high-molecular-weight P3HT, which do not change significantly after annealing at 150°C up to 30 min.

Platinum blue staining of cells grown in electrospun scaffolds.

PubMed

Yusuf, Mohammed; Millas, Ana Luiza G; Estandarte, Ana Katrina C; Bhella, Gurdeep K; McKean, Robert; Bittencourt, Edison; Robinson, Ian K

2014-01-01

Fibroblast cells grown in electrospun polymer scaffolds were stained with platinum blue, a heavy metal stain, and imaged using scanning electron microscopy. Good contrast on the cells was achieved compared with samples that were gold sputter coated. The cell morphology could be clearly observed, and the cells could be distinguished from the scaffold fibers. Here we optimized the required concentration of platinum blue for imaging cells grown in scaffolds and show that a higher concentration causes platinum aggregation. Overall, platinum blue is a useful stain for imaging cells because of its enhanced contrast using scanning electron microscopy (SEM). In the future it would be useful to investigate cell growth and morphology using three-dimensional imaging methods.

Analysis of liquid suspensions using scanning electron microscopy in transmission: estimation of the water film thickness using Monte-Carlo simulations.

PubMed

Xiao, J; Foray, G; Masenelli-Varlot, K

2018-02-01

Environmental scanning electron microscopy (ESEM) allows the observation of liquids under specific conditions of pressure and temperature. Moreover, when working in the transmission mode, that is in scanning transmission electron microscopy (STEM), nano-objects can be analysed inside a liquid. The contrast in the images is mass-thickness dependent as in STEM-in-TEM (transmission electron microscopy) using closed cells. However, in STEM-in-ESEM, as the liquid-vapour equilibrium is kept dynamically, the thickness of the water droplet remains unknown. In this paper, the contrasts measured in the experimental images are compared with calculations using Monte-Carlo simulations in order to estimate the thickness of water. Two examples are given. On gold nanoparticles, the thickness of a thick film can be estimated thanks to a contrast inversion. On core-shell latex particles, the grey level of the shell compared with those of the core and of the water film gives a relatively precise measurement of the water film thickness. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Two-dimensional confocal laser scanning microscopy image correlation for nanoparticle flow velocimetry

NASA Astrophysics Data System (ADS)

Jun, Brian; Giarra, Matthew; Golz, Brian; Main, Russell; Vlachos, Pavlos

2016-11-01

We present a methodology to mitigate the major sources of error associated with two-dimensional confocal laser scanning microscopy (CLSM) images of nanoparticles flowing through a microfluidic channel. The correlation-based velocity measurements from CLSM images are subject to random error due to the Brownian motion of nanometer-sized tracer particles, and a bias error due to the formation of images by raster scanning. Here, we develop a novel ensemble phase correlation with dynamic optimal filter that maximizes the correlation strength, which diminishes the random error. In addition, we introduce an analytical model of CLSM measurement bias error correction due to two-dimensional image scanning of tracer particles. We tested our technique using both synthetic and experimental images of nanoparticles flowing through a microfluidic channel. We observed that our technique reduced the error by up to a factor of ten compared to ensemble standard cross correlation (SCC) for the images tested in the present work. Subsequently, we will assess our framework further, by interrogating nanoscale flow in the cell culture environment (transport within the lacunar-canalicular system) to demonstrate our ability to accurately resolve flow measurements in a biological system.

Multiphoton imaging with high peak power VECSELs

NASA Astrophysics Data System (ADS)

Mirkhanov, Shamil; Quarterman, Adrian H.; Swift, Samuel; Praveen, Bavishna B.; Smyth, Conor J. C.; Wilcox, Keith G.

2016-03-01

Multiphoton imaging (MMPI) has become one of thee key non-invasive light microscopy techniques. This technique allows deep tissue imaging with high resolution and less photo-damage than conventional confocal microscopy. MPI is type of laser-scanning microscopy that employs localized nonlinear excitation, so that fluorescence is excited only with is scanned focal volume. For many years, Ti: sapphire femtosecond lasers have been the leading light sources for MPI applications. However, recent developments in laser sources and new types of fluorophores indicate that longer wavelength excitation could be a good alternative for these applications. Mode-locked VECSEELs have the potential to be low cost, compact light sources for MPI systems, with the additional advantage of broad wavelength coverage through use of different semiconductor material systems. Here, we use a femtosecond fibber laser to investigate the effect average power and repetition rate has on MPI image quality, to allow us to optimize our mode-locked VVECSELs for MPI.

Rapid spontaneous Raman light sheet microscopy using cw-lasers and tunable filters

PubMed Central

Rocha-Mendoza, Israel; Licea-Rodriguez, Jacob; Marro, Mónica; Olarte, Omar E.; Plata-Sanchez, Marcos; Loza-Alvarez, Pablo

2015-01-01

We perform rapid spontaneous Raman 2D imaging in light-sheet microscopy using continuous wave lasers and interferometric tunable filters. By angularly tuning the filter, the cut-on/off edge transitions are scanned along the excited Stokes wavelengths. This allows obtaining cumulative intensity profiles of the scanned vibrational bands, which are recorded on image stacks; resembling a spectral version of the knife-edge technique to measure intensity profiles. A further differentiation of the stack retrieves the Raman spectra at each pixel of the image which inherits the 3D resolution of the host light sheet system. We demonstrate this technique using solvent solutions and composites of polystyrene beads and lipid droplets immersed in agar and by imaging the C–H (2800-3100cm−1) region in a C. elegans worm. The image acquisition time results in 4 orders of magnitude faster than confocal point scanning Raman systems, allowing the possibility of performing fast spontaneous Raman·3D-imaging on biological samples. PMID:26417514

Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging

PubMed Central

Thériault, Gabrielle; Cottet, Martin; Castonguay, Annie; McCarthy, Nathalie; De Koninck, Yves

2014-01-01

Two-photon microscopy has revolutionized functional cellular imaging in tissue, but although the highly confined depth of field (DOF) of standard set-ups yields great optical sectioning, it also limits imaging speed in volume samples and ease of use. For this reason, we recently presented a simple and retrofittable modification to the two-photon laser-scanning microscope which extends the DOF through the use of an axicon (conical lens). Here we demonstrate three significant benefits of this technique using biological samples commonly employed in the field of neuroscience. First, we use a sample of neurons grown in culture and move it along the z-axis, showing that a more stable focus is achieved without compromise on transverse resolution. Second, we monitor 3D population dynamics in an acute slice of live mouse cortex, demonstrating that faster volumetric scans can be conducted. Third, we acquire a stereoscopic image of neurons and their dendrites in a fixed sample of mouse cortex, using only two scans instead of the complete stack and calculations required by standard systems. Taken together, these advantages, combined with the ease of integration into pre-existing systems, make the extended depth-of-field imaging based on Bessel beams a strong asset for the field of microscopy and life sciences in general. PMID:24904284

Gold Nanoparticle Quantitation by Whole Cell Tomography.

PubMed

Sanders, Aric W; Jeerage, Kavita M; Schwartz, Cindi L; Curtin, Alexandra E; Chiaramonti, Ann N

2015-12-22

Many proposed biomedical applications for engineered gold nanoparticles require their incorporation by mammalian cells in specific numbers and locations. Here, the number of gold nanoparticles inside of individual mammalian stem cells was characterized using fast focused ion beam-scanning electron microscopy based tomography. Enhanced optical microscopy was used to provide a multiscale map of the in vitro sample, which allows cells of interest to be identified within their local environment. Cells were then serially sectioned using a gallium ion beam and imaged using a scanning electron beam. To confirm the accuracy of single cross sections, nanoparticles in similar cross sections were imaged using transmission electron microscopy and scanning helium ion microscopy. Complete tomographic series were then used to count the nanoparticles inside of each cell and measure their spatial distribution. We investigated the influence of slice thickness on counting single particles and clusters as well as nanoparticle packing within clusters. For 60 nm citrate stabilized particles, the nanoparticle cluster packing volume is 2.15 ± 0.20 times the volume of the bare gold nanoparticles.

Coherent x-ray zoom condenser lens for diffractive and scanning microscopy.

PubMed

Kimura, Takashi; Matsuyama, Satoshi; Yamauchi, Kazuto; Nishino, Yoshinori

2013-04-22

We propose a coherent x-ray zoom condenser lens composed of two-stage deformable Kirkpatrick-Baez mirrors. The lens delivers coherent x-rays with a controllable beam size, from one micrometer to a few tens of nanometers, at a fixed focal position. The lens is suitable for diffractive and scanning microscopy. We also propose non-scanning coherent diffraction microscopy for extended objects by using an apodized focused beam produced by the lens with a spatial filter. The proposed apodized-illumination method will be useful in highly efficient imaging with ultimate storage ring sources, and will also open the way to single-shot coherent diffraction microscopy of extended objects with x-ray free-electron lasers.

Fully Hydrated Yeast Cells Imaged with Electron Microscopy

PubMed Central

Peckys, Diana B.; Mazur, Peter; Gould, Kathleen L.; de Jonge, Niels

2011-01-01

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccaromyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. PMID:21575587

Fully hydrated yeast cells imaged with electron microscopy.

PubMed

Peckys, Diana B; Mazur, Peter; Gould, Kathleen L; de Jonge, Niels

2011-05-18

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccharomyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

High-resolution mapping of molecules in an ionic liquid via scanning transmission electron microscopy.

PubMed

Miyata, Tomohiro; Mizoguchi, Teruyasu

2018-03-01

Understanding structures and spatial distributions of molecules in liquid phases is crucial for the control of liquid properties and to develop efficient liquid-phase processes. Here, real-space mapping of molecular distributions in a liquid was performed. Specifically, the ionic liquid 1-Ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (C2mimTFSI) was imaged using atomic-resolution scanning transmission electron microscopy. Simulations revealed network-like bright regions in the images that were attributed to the TFSI- anion, with minimal contributions from the C2mim+ cation. Simple visualization of the TFSI- distribution in the liquid sample was achieved by binarizing the experimental image.

Carbon contamination in scanning transmission electron microscopy and its impact on phase-plate applications.

PubMed

Hettler, Simon; Dries, Manuel; Hermann, Peter; Obermair, Martin; Gerthsen, Dagmar; Malac, Marek

2017-05-01

We analyze electron-beam induced carbon contamination in a transmission electron microscope. The study is performed on thin films potentially suitable as phase plates for phase-contrast transmission electron microscopy. Electron energy-loss spectroscopy and phase-plate imaging is utilized to analyze the contamination. The deposited contamination layer is identified as a graphitic carbon layer which is not prone to electrostatic charging whereas a non-conductive underlying substrate charges. Several methods that inhibit contamination are evaluated and the impact of carbon contamination on phase-plate imaging is discussed. The findings are in general interesting for scanning transmission electron microscopy applications. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

High-Precision Photothermal Ablation Using Biocompatible Palladium Nanoparticles and Laser Scanning Microscopy

PubMed Central

2018-01-01

Herein, we report a straightforward method for the scalable preparation of Pd nanoparticles (Pd-NPs) with reduced inherent cytotoxicity and high photothermal conversion capacity. These Pd-NPs are rapidly taken up by cells and able to kill labeled cancer cells upon short exposure to near-infrared (NIR) light. Following cell treatment with Pd-NPs, ablated areas were patterned with high precision by laser scanning microscopy, allowing one to perform cell migration assays with unprecedented accuracy. Using coherent Raman microscopy, cells containing Pd-NPs were simultaneously ablated and imaged. This novel methodology was combined with intravital imaging to mediate microablation of cancerous tissue in tumor xenografts in mice. PMID:29320154

Cement paste surface roughness analysis using coherence scanning interferometry and confocal microscopy

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

Apedo, K.L., E-mail: apedo@unistra.fr; Munzer, C.; He, H.

2015-02-15

Scanning electron microscopy and scanning probe microscopy have been used for several decades to better understand the microstructure of cementitious materials. Very limited work has been performed to date to study the roughness of cementitious materials by optical microscopy such as coherence scanning interferometry (CSI) and chromatic confocal sensing (CCS). The objective of this paper is to better understand how CSI can be used as a tool to analyze surface roughness and topography of cement pastes. Observations from a series of images acquired using this technique on both polished and unpolished samples are described. The results from CSI are comparedmore » with those from a STIL confocal microscopy technique (SCM). Comparison between both optical techniques demonstrates the ability of CSI to measure both polished and unpolished cement pastes. - Highlights: • Coherence scanning interferometry (CSI) was used to analyze cement paste surfaces. • The results from the CSI were compared with those from a confocal microscopy. • 3D roughness parameters were obtained using the window resizing method. • Polished and unpolished cement pastes were studied.« less

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain.

PubMed

Kuipers, Jeroen; Kalicharan, Ruby D; Wolters, Anouk H G; van Ham, Tjakko J; Giepmans, Ben N G

2016-05-25

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae(1-7). Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture(1-5). Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)(8) on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

PubMed Central

Kuipers, Jeroen; Kalicharan, Ruby D.; Wolters, Anouk H. G.

2016-01-01

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner. PMID:27285162

Wavelength scanning achieves pixel super-resolution in holographic on-chip microscopy

NASA Astrophysics Data System (ADS)

Luo, Wei; Göröcs, Zoltan; Zhang, Yibo; Feizi, Alborz; Greenbaum, Alon; Ozcan, Aydogan

2016-03-01

Lensfree holographic on-chip imaging is a potent solution for high-resolution and field-portable bright-field imaging over a wide field-of-view. Previous lensfree imaging approaches utilize a pixel super-resolution technique, which relies on sub-pixel lateral displacements between the lensfree diffraction patterns and the image sensor's pixel-array, to achieve sub-micron resolution under unit magnification using state-of-the-art CMOS imager chips, commonly used in e.g., mobile-phones. Here we report, for the first time, a wavelength scanning based pixel super-resolution technique in lensfree holographic imaging. We developed an iterative super-resolution algorithm, which generates high-resolution reconstructions of the specimen from low-resolution (i.e., under-sampled) diffraction patterns recorded at multiple wavelengths within a narrow spectral range (e.g., 10-30 nm). Compared with lateral shift-based pixel super-resolution, this wavelength scanning approach does not require any physical shifts in the imaging setup, and the resolution improvement is uniform in all directions across the sensor-array. Our wavelength scanning super-resolution approach can also be integrated with multi-height and/or multi-angle on-chip imaging techniques to obtain even higher resolution reconstructions. For example, using wavelength scanning together with multi-angle illumination, we achieved a halfpitch resolution of 250 nm, corresponding to a numerical aperture of 1. In addition to pixel super-resolution, the small scanning steps in wavelength also enable us to robustly unwrap phase, revealing the specimen's optical path length in our reconstructed images. We believe that this new wavelength scanning based pixel super-resolution approach can provide competitive microscopy solutions for high-resolution and field-portable imaging needs, potentially impacting tele-pathology applications in resource-limited-settings.

Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy

PubMed Central

Vélez-Ortega, A. Catalina; Frolenkov, Gregory I.

2016-01-01

The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3 to 4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette –which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier– is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface. Here we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations. PMID:27259929

Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy.

PubMed

Vélez-Ortega, A Catalina; Frolenkov, Gregory I

2016-01-01

The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3-4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette-which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier-is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface.Here, we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations.

High-resolution measurements of the multilayer ultra-structure of articular cartilage and their translational potential

PubMed Central

2014-01-01

Current musculoskeletal imaging techniques usually target the macro-morphology of articular cartilage or use histological analysis. These techniques are able to reveal advanced osteoarthritic changes in articular cartilage but fail to give detailed information to distinguish early osteoarthritis from healthy cartilage, and this necessitates high-resolution imaging techniques measuring cells and the extracellular matrix within the multilayer structure of articular cartilage. This review provides a comprehensive exploration of the cellular components and extracellular matrix of articular cartilage as well as high-resolution imaging techniques, including magnetic resonance image, electron microscopy, confocal laser scanning microscopy, second harmonic generation microscopy, and laser scanning confocal arthroscopy, in the measurement of multilayer ultra-structures of articular cartilage. This review also provides an overview for micro-structural analysis of the main components of normal or osteoarthritic cartilage and discusses the potential and challenges associated with developing non-invasive high-resolution imaging techniques for both research and clinical diagnosis of early to late osteoarthritis. PMID:24946278

Optical imaging beyond the diffraction limit by SNEM: effects of AFM tip modifications with thiol monolayers on imaging quality.

PubMed

Cumurcu, Aysegul; Diaz, Jordi; Lindsay, Ian D; de Beer, Sissi; Duvigneau, Joost; Schön, Peter; Julius Vancso, G

2015-03-01

Tip-enhanced nanoscale optical imaging techniques such as apertureless scanning near-field optical microscopy (a-SNOM) and scanning near-field ellipsometric microscopy (SNEM) applications can suffer from a steady degradation in performance due to adhesion of atmospheric contaminants to the metal coated tip. Here, we demonstrate that a self-assembled monolayer (SAM) of ethanethiol (EtSH) is an effective means of protecting gold-coated atomic force microscopy (AFM) probe tips from accumulation of surface contaminants during prolonged exposure to ambient air. The period over which they yield consistent and reproducible results for scanning near-field ellipsometric microscopy (SNEM) imaging is thus extended. SNEM optical images of a microphase separated polystyrene-block-poly (methylmethacrylate) (PS-b-PMMA) diblock copolymer film, which were captured with bare and SAM-protected gold-coated AFM probes, both immediately after coating and following five days of storage in ambient air, were compared. During this period the intensity of the optical signals from the untreated gold tip fell by 66%, while those from the SAM protected tip fell by 14%. Additionally, gold coated AFM probe tips were modified with various lengths of alkanethiols to measure the change in intensity variation in the optical images with SAM layer thickness. The experimental results were compared to point dipole model calculations. While a SAM of 1-dodecanethiol (DoSH) was found to strongly suppress field enhancement we find that it can be locally removed from the tip apex by deforming the molecules under load, restoring SNEM image contrast. Copyright © 2014 Elsevier B.V. All rights reserved.

Quantitative 3D comparison of biofilm imaged by X-ray micro-tomography and two-photon laser scanning microscopy.

PubMed

Larue, A E; Swider, P; Duru, P; Daviaud, D; Quintard, M; Davit, Y

2018-06-21

Optical imaging techniques for biofilm observation, like laser scanning microscopy, are not applicable when investigating biofilm formation in opaque porous media. X-ray micro-tomography (X-ray CMT) might be an alternative but it finds limitations in similarity of X-ray absorption coefficients for the biofilm and aqueous phases. To overcome this difficulty, barium sulphate was used in Davit et al. (2011) to enable high-resolution 3D imaging of biofilm via X-ray CMT. However, this approach lacks comparison with well-established imaging methods, which are known to capture the fine structures of biofilms, as well as uncertainty quantification. Here, we compare two-photon laser scanning microscopy (TPLSM) images of Pseudomonas Aeruginosa biofilm grown in glass capillaries against X-ray CMT using an improved protocol where barium sulphate is combined with low-gelling temperature agarose to avoid sedimentation. Calibrated phantoms consisting of mono-dispersed fluorescent and X-ray absorbent beads were used to evaluate the uncertainty associated with our protocol along with three different segmentation techniques, namely hysteresis, watershed and region growing, to determine the bias relative to image binarization. Metrics such as volume, 3D surface area and thickness were measured and comparison of both imaging modalities shows that X-ray CMT of biofilm using our protocol yields an accuracy that is comparable and even better in certain respects than TPLSM, even in a nonporous system that is largely favourable to TPLSM. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Reprint of: Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film.

PubMed

Nishiyama, Hidetoshi; Suga, Mitsuo; Ogura, Toshihiko; Maruyama, Yuusuke; Koizumi, Mitsuru; Mio, Kazuhiro; Kitamura, Shinichi; Sato, Chikara

2010-11-01

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry. Copyright © 2010 Elsevier Inc. All rights reserved.

Water-Immersible MEMS scanning mirror designed for wide-field fast-scanning photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Yao, Junjie; Huang, Chih-Hsien; Martel, Catherine; Maslov, Konstantin I.; Wang, Lidai; Yang, Joon-Mo; Gao, Liang; Randolph, Gwendalyn; Zou, Jun; Wang, Lihong V.

2013-03-01

By offering images with high spatial resolution and unique optical absorption contrast, optical-resolution photoacoustic microscopy (OR-PAM) has gained increasing attention in biomedical research. Recent developments in OR-PAM have improved its imaging speed, but have sacrificed either the detection sensitivity or field of view or both. We have developed a wide-field fast-scanning OR-PAM by using a water-immersible MEMS scanning mirror (MEMS-ORPAM). Made of silicon with a gold coating, the MEMS mirror plate can reflect both optical and acoustic beams. Because it uses an electromagnetic driving force, the whole MEMS scanning system can be submerged in water. In MEMS-ORPAM, the optical and acoustic beams are confocally configured and simultaneously steered, which ensures uniform detection sensitivity. A B-scan imaging speed as high as 400 Hz can be achieved over a 3 mm scanning range. A diffraction-limited lateral resolution of 2.4 μm in water and a maximum imaging depth of 1.1 mm in soft tissue have been experimentally determined. Using the system, we imaged the flow dynamics of both red blood cells and carbon particles in a mouse ear in vivo. By using Evans blue dye as the contrast agent, we also imaged the flow dynamics of lymphatic vessels in a mouse tail in vivo. The results show that MEMS-OR-PAM could be a powerful tool for studying highly dynamic and time-sensitive biological phenomena.

Method for imaging liquid and dielectric materials with scanning polarization force microscopy

DOEpatents

Hu, Jun; Ogletree, D. Frank; Salmeron, Miguel; Xiao, Xudong

1999-01-01

The invention images dielectric polarization forces on surfaces induced by a charged scanning force microscope (SFM) probe tip. On insulators, the major contribution to the surface polarizability at low frequencies is from surface ions. The mobility of these ions depends strongly on the humidity. Using the inventive SFM, liquid films, droplets, and other weakly adsorbed materials have been imaged.

Apparatus for imaging liquid and dielectric materials with scanning polarization force microscopy

DOEpatents

Hu, Jun; Ogletree, D. Frank; Salmeron, Miguel; Xiao, Xudong

1998-01-01

The invention images dielectric polarization forces on surfaces induced by a charged scanning force microscope (SFM) probe tip. On insulators, the major contribution to the surface polarizability at low frequencies is from surface ions. The mobility of these ions depends strongly on the humidity. Using the inventive SFM, liquid films, droplets, and other weakly adsorbed materials have been imaged.

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.

Performance of a three-dimensional-printed microscanner in a laser scanning microscopy application

NASA Astrophysics Data System (ADS)

Oyman, Hilmi Artun; Gokdel, Yigit Daghan; Ferhanoglu, Onur; Yalcinkaya, Arda Deniz

2018-04-01

A magnetically actuated microscanner is used in a laser scanning microscopy application. Stress distribution along the circular-profiled flexure is compared with a rectangular counterpart in finite-element environment. Magnetic actuation mechanism of the scanning unit is explained in detail. Moreover, reliability of the scanner is tested for 3×106 cycle. The scanning device is designed to meet a confocal microscopy application providing 100 μm×100 μm field of view and <3-μm lateral resolution. The resonance frequencies of the device were analytically modeled, where we obtained 130- and 268-Hz resonance values for the out-of-plane and torsion modes, respectively. The scanning device provided an optical scan angle about 2.5 deg for 170-mA drive current, enabling the desired field of view for our custom built confocal microscope setup. Finally, imaging experiments were conducted on a resolution target, showcasing the desired scan area and resolution.

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

PubMed Central

Pérez-Alvarez, Alberto; Araque, Alfonso; Martín, Eduardo D.

2013-01-01

In vivo imaging is one of the ultimate and fundamental approaches for the study of the brain. Two-photon laser scanning microscopy (2PLSM) constitutes the state-of-the-art technique in current neuroscience to address questions regarding brain cell structure, development and function, blood flow regulation and metabolism. This technique evolved from laser scanning confocal microscopy (LSCM), which impacted the field with a major improvement in image resolution of live tissues in the 1980s compared to widefield microscopy. While nowadays some of the unparalleled features of 2PLSM make it the tool of choice for brain studies in vivo, such as the possibility to image deep within a tissue, LSCM can still be useful in this matter. Here we discuss the validity and limitations of LSCM and provide a guide to perform high-resolution in vivo imaging of the brain of live rodents with minimal mechanical disruption employing LSCM. We describe the surgical procedure and experimental setup that allowed us to record intracellular calcium variations in astrocytes evoked by sensory stimulation, and to monitor intact neuronal dendritic spines and astrocytic processes as well as blood vessel dynamics. Therefore, in spite of certain limitations that need to be carefully considered, LSCM constitutes a useful, convenient, and affordable tool for brain studies in vivo. PMID:23658537

Design of a handheld optical coherence microscopy endoscope

NASA Astrophysics Data System (ADS)

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

2011-06-01

Optical coherence microscopy (OCM) combines coherence gating, high numerical aperture optics, and a fiber-core pinhole to provide high axial and lateral resolution with relatively large depth of imaging. We present a handheld rigid OCM endoscope designed for small animal surgical imaging, with a 6-mm diam tip, 1-mm scan width, and 1-mm imaging depth. X-Y scanning is performed distally with mirrors mounted to micro galvonometer scanners incorporated into the endoscope handle. The endoscope optical design consists of scanning doublets, an afocal Hopkins relay lens system, a 0.4 numerical aperture water immersion objective, and a cover glass. This endoscope can resolve laterally a 1.4-μm line pair feature and has an axial resolution (full width half maximum) of 5.4 μm. Images taken with this endoscope of fresh ex-vivo mouse ovaries show structural features, such as corpus luteum, primary follicles, growing follicles, and fallopian tubes. This rigid handheld OCM endoscope can be useful for a variety of minimally invasive and surgical imaging applications.

Imaging ballistic carrier trajectories in graphene using scanning gate microscopy

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

Morikawa, Sei; Masubuchi, Satoru; Dou, Ziwei

2015-12-14

We use scanning gate microscopy to map out the trajectories of ballistic carriers in high-mobility graphene encapsulated by hexagonal boron nitride and subject to a weak magnetic field. We employ a magnetic focusing geometry to image carriers that emerge ballistically from an injector, follow a cyclotron path due to the Lorentz force from an applied magnetic field, and land on an adjacent collector probe. The local electric field generated by the scanning tip in the vicinity of the carriers deflects their trajectories, modifying the proportion of carriers focused into the collector. By measuring the voltage at the collector while scanningmore » the tip, we are able to obtain images with arcs that are consistent with the expected cyclotron motion. We also demonstrate that the tip can be used to redirect misaligned carriers back to the collector.« less

A line scanned light-sheet microscope with phase shaped self-reconstructing beams.

PubMed

Fahrbach, Florian O; Rohrbach, Alexander

2010-11-08

We recently demonstrated that Microscopy with Self-Reconstructing Beams (MISERB) increases both image quality and penetration depth of illumination beams in strongly scattering media. Based on the concept of line scanned light-sheet microscopy, we present an add-on module to a standard inverted microscope using a scanned beam that is shaped in phase and amplitude by a spatial light modulator. We explain technical details of the setup as well as of the holograms for the creation, positioning and scaling of static light-sheets, Gaussian beams and Bessel beams. The comparison of images from identical sample areas illuminated by different beams allows a precise assessment of the interconnection between beam shape and image quality. The superior propagation ability of Bessel beams through inhomogeneous media is demonstrated by measurements on various scattering media.

Deep ultraviolet scanning near-field optical microscopy for the structural analysis of organic and biological materials

NASA Astrophysics Data System (ADS)

Aoki, Hiroyuki; Hamamatsu, Toyohiro; Ito, Shinzaburo

2004-01-01

Scanning near-field optical microscopy (SNOM) using a deep ultraviolet (DUV) light source was developed for in situ imaging of a variety of chemical species without staining. Numerous kinds of chemical species have a carbon-carbon double bond or aromatic group in their chemical structure, which can be excited at the wavelength below 300 nm. In this study, the wavelength range available for SNOM imaging was extended to the DUV region. DUV-SNOM allowed the direct imaging of polymer thin films with high detection sensitivity and spatial resolution of several tens of nanometers. In addition to the polymer materials, we demonstrated the near-field imaging of a cell without using a fluorescence label.

Analysing magnetism using scanning SQUID microscopy.

PubMed

Reith, P; Renshaw Wang, X; Hilgenkamp, H

2017-12-01

Scanning superconducting quantum interference device microscopy (SSM) is a scanning probe technique that images local magnetic flux, which allows for mapping of magnetic fields with high field and spatial accuracy. Many studies involving SSM have been published in the last few decades, using SSM to make qualitative statements about magnetism. However, quantitative analysis using SSM has received less attention. In this work, we discuss several aspects of interpreting SSM images and methods to improve quantitative analysis. First, we analyse the spatial resolution and how it depends on several factors. Second, we discuss the analysis of SSM scans and the information obtained from the SSM data. Using simulations, we show how signals evolve as a function of changing scan height, SQUID loop size, magnetization strength, and orientation. We also investigated 2-dimensional autocorrelation analysis to extract information about the size, shape, and symmetry of magnetic features. Finally, we provide an outlook on possible future applications and improvements.

Analysing magnetism using scanning SQUID microscopy

NASA Astrophysics Data System (ADS)

Reith, P.; Renshaw Wang, X.; Hilgenkamp, H.

2017-12-01

Scanning superconducting quantum interference device microscopy (SSM) is a scanning probe technique that images local magnetic flux, which allows for mapping of magnetic fields with high field and spatial accuracy. Many studies involving SSM have been published in the last few decades, using SSM to make qualitative statements about magnetism. However, quantitative analysis using SSM has received less attention. In this work, we discuss several aspects of interpreting SSM images and methods to improve quantitative analysis. First, we analyse the spatial resolution and how it depends on several factors. Second, we discuss the analysis of SSM scans and the information obtained from the SSM data. Using simulations, we show how signals evolve as a function of changing scan height, SQUID loop size, magnetization strength, and orientation. We also investigated 2-dimensional autocorrelation analysis to extract information about the size, shape, and symmetry of magnetic features. Finally, we provide an outlook on possible future applications and improvements.

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

PubMed Central

Flaberg, Emilie; Sabelström, Per; Strandh, Christer; Szekely, Laszlo

2008-01-01

Background Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. Methods Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). Results We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. Conclusion The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes. PMID:18627634

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

PubMed

Flaberg, Emilie; Sabelström, Per; Strandh, Christer; Szekely, Laszlo

2008-07-16

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

Nanoscale Imaging of Buried Structures via Scanning Near-Field Ultrasound Holography

NASA Astrophysics Data System (ADS)

Shekhawat, Gajendra S.; Dravid, Vinayak P.

2005-10-01

A nondestructive imaging method, scanning near-field ultrasound holography (SNFUH), has been developed that provides depth information as well as spatial resolution at the 10- to 100-nanometer scale. In SNFUH, the phase and amplitude of the scattered specimen ultrasound wave, reflected in perturbation to the surface acoustic standing wave, are mapped with a scanning probe microscopy platform to provide nanoscale-resolution images of the internal substructure of diverse materials. We have used SNFUH to image buried nanostructures, to perform subsurface metrology in microelectronic structures, and to image malaria parasites in red blood cells.

Nonlinear Focal Modulation Microscopy.

PubMed

Zhao, Guangyuan; Zheng, Cheng; Kuang, Cuifang; Zhou, Renjie; Kabir, Mohammad M; Toussaint, Kimani C; Wang, Wensheng; Xu, Liang; Li, Haifeng; Xiu, Peng; Liu, Xu

2018-05-11

We demonstrate nonlinear focal modulation microscopy (NFOMM) to achieve superresolution imaging. Traditional approaches to superresolution that utilize point scanning often rely on spatially reducing the size of the emission pattern by directly narrowing (e.g., through minimizing the detection pinhole in Airyscan, Zeiss) or indirectly peeling its outer profiles [e.g., through depleting the outer emission region in stimulated emission depletion (STED) microscopy]. We show that an alternative conceptualization that focuses on maximizing the optical system's frequency shifting ability offers advantages in further improving resolution while reducing system complexity. In NFOMM, a spatial light modulator and a suitably intense laser illumination are used to implement nonlinear focal-field modulation to achieve a transverse spatial resolution of ∼60  nm (∼λ/10). We show that NFOMM is comparable with STED microscopy and suitable for fundamental biology studies, as evidenced in imaging nuclear pore complexes, tubulin and vimentin in Vero cells. Since NFOMM is readily implemented as an add-on module to a laser-scanning microscope, we anticipate wide utility of this new imaging technique.

Nonlinear Focal Modulation Microscopy

NASA Astrophysics Data System (ADS)

Zhao, Guangyuan; Zheng, Cheng; Kuang, Cuifang; Zhou, Renjie; Kabir, Mohammad M.; Toussaint, Kimani C.; Wang, Wensheng; Xu, Liang; Li, Haifeng; Xiu, Peng; Liu, Xu

2018-05-01

We demonstrate nonlinear focal modulation microscopy (NFOMM) to achieve superresolution imaging. Traditional approaches to superresolution that utilize point scanning often rely on spatially reducing the size of the emission pattern by directly narrowing (e.g., through minimizing the detection pinhole in Airyscan, Zeiss) or indirectly peeling its outer profiles [e.g., through depleting the outer emission region in stimulated emission depletion (STED) microscopy]. We show that an alternative conceptualization that focuses on maximizing the optical system's frequency shifting ability offers advantages in further improving resolution while reducing system complexity. In NFOMM, a spatial light modulator and a suitably intense laser illumination are used to implement nonlinear focal-field modulation to achieve a transverse spatial resolution of ˜60 nm (˜λ /10 ). We show that NFOMM is comparable with STED microscopy and suitable for fundamental biology studies, as evidenced in imaging nuclear pore complexes, tubulin and vimentin in Vero cells. Since NFOMM is readily implemented as an add-on module to a laser-scanning microscope, we anticipate wide utility of this new imaging technique.

Imaging interactions of metal oxide nanoparticles with macrophage cells by ultra-high resolution scanning electron microscopy techniques.

PubMed

Plascencia-Villa, Germán; Starr, Clarise R; Armstrong, Linda S; Ponce, Arturo; José-Yacamán, Miguel

2012-11-01

Use of engineered metal oxide nanoparticles in a plethora of biological applications and custom products has warned about some possible dose-dependent cytotoxic effects. Macrophages are key components of the innate immune system used to study possible toxic effects and internalization of different nanoparticulate materials. In this work, ultra-high resolution field emission scanning electron microscopy (FE-SEM) was used to offer new insights into the dynamical processes of interaction of nanomaterials with macrophage cells dosed with different concentrations of metal oxide nanoparticles (CeO(2), TiO(2) and ZnO). The versatility of FE-SEM has allowed obtaining a detailed characterization of processes of adsorption and endocytosis of nanoparticles, by using advanced analytical and imaging techniques on complete unstained uncoated cells, including secondary electron imaging, high-sensitive backscattered electron imaging, X-ray microanalysis and stereoimaging. Low voltage BF/DF-STEM confirmed nanoparticle adsorption and internalization into endosomes of CeO(2) and TiO(2), whereas ZnO develop apoptosis after 24 h of interaction caused by dissolution and invasion of cell nucleus. Ultra-high resolution scanning electron microscopy techniques provided new insights into interactions of inorganic nanoparticles with macrophage cells with high spatial resolution.

Simultaneous noncontact topography and electrochemical imaging by SECM/SICM featuring ion current feedback regulation.

PubMed

Takahashi, Yasufumi; Shevchuk, Andrew I; Novak, Pavel; Murakami, Yumi; Shiku, Hitoshi; Korchev, Yuri E; Matsue, Tomokazu

2010-07-28

We described a hybrid system of scanning electrochemical microscopy (SECM) and scanning ion conductance microscopy (SICM) with ion current feedback nanopositioning control for simultaneous imaging of noncontact topography and spatial distribution of electrochemical species. A nanopipette/nanoring electrode probe provided submicrometer resolution of the electrochemical measurement on surfaces with complex topology. The SECM/SICM probe had an aperture radius of 220 nm. The inner and outer radii of the SECM Au nanoring electrode were 330 and 550 nm, respectively. Characterization of the probe was performed with scanning electron microscopy (SEM), cyclic voltammetry (CV), and approach curve measurements. SECM/SICM was applied to simultaneous imaging of topography and electrochemical responses of enzymes (horse radish peroxidase (HRP) and glucose oxidase (GOD)) and single live cells (A6 cells, superior cervical ganglion (SCG) cells, and cardiac myocytes). The measurements revealed the distribution of activity of the enzyme spots on uneven surfaces with submicrometer resolution. SECM/SICM acquired high resolution topographic images of cells together with the map of electrochemical signals. This combined technique was also applied to the evaluation of the permeation property of electroactive species through cellular membranes.

Computational modeling of optical projection tomographic microscopy using the finite difference time domain method.

PubMed

Coe, Ryan L; Seibel, Eric J

2012-12-01

We present a method for modeling image formation in optical projection tomographic microscopy (OPTM) using high numerical aperture (NA) condensers and objectives. Similar to techniques used in computed tomography, OPTM produces three-dimensional, reconstructed images of single cells from two-dimensional projections. The model is capable of simulating axial scanning of a microscope objective to produce projections, which are reconstructed using filtered backprojection. Simulation of optical scattering in transmission optical microscopy is designed to analyze all aspects of OPTM image formation, such as degree of specimen staining, refractive-index matching, and objective scanning. In this preliminary work, a set of simulations is performed to examine the effect of changing the condenser NA, objective scan range, and complex refractive index on the final reconstruction of a microshell with an outer radius of 1.5 μm and an inner radius of 0.9 μm. The model lays the groundwork for optimizing OPTM imaging parameters and triaging efforts to further improve the overall system design. As the model is expanded in the future, it will be used to simulate a more realistic cell, which could lead to even greater impact.

Nanoscale cellular imaging with scanning angle interference microscopy.

PubMed

DuFort, Christopher; Paszek, Matthew

2014-01-01

Fluorescence microscopy is among the most widely utilized tools in cell and molecular biology due to its ability to noninvasively obtain time-resolved images of live cells with molecule-specific contrast. In this chapter, we describe a simple high-resolution technique, scanning angle interference microscopy (SAIM), for the imaging and localization of fluorescent molecules with nanometer precision along the optical axis. In SAIM, samples above a reflective surface are sequentially scanned with an excitation laser at varying angles of incidence. Interference patterns generated between the incident and reflected lights result in an emission intensity that depends on the height of a fluorophore above the silicon surface and the angle of the incident radiation. The measured fluorescence intensities are then fit to an optical model to localize the labeled molecules along the z-axis with 5-10 nm precision and diffraction-limited lateral resolution. SAIM is easily implemented on widely available commercial total internal reflection fluorescence microscopes, offering potential for widespread use in cell biology. Here, we describe the setup of SAIM and its application for imaging cellular structures near (<1 μm) the sample substrate. © 2014 Elsevier Inc. All rights reserved.

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

PubMed

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

2015-07-01

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

High Resolution Helium Ion Scanning Microscopy of the Rat Kidney

PubMed Central

Rice, William L.; Van Hoek, Alfred N.; Păunescu, Teodor G.; Huynh, Chuong; Goetze, Bernhard; Singh, Bipin; Scipioni, Larry; Stern, Lewis A.; Brown, Dennis

2013-01-01

Helium ion scanning microscopy is a novel imaging technology with the potential to provide sub-nanometer resolution images of uncoated biological tissues. So far, however, it has been used mainly in materials science applications. Here, we took advantage of helium ion microscopy to explore the epithelium of the rat kidney with unsurpassed image quality and detail. In addition, we evaluated different tissue preparation methods for their ability to preserve tissue architecture. We found that high contrast, high resolution imaging of the renal tubule surface is possible with a relatively simple processing procedure that consists of transcardial perfusion with aldehyde fixatives, vibratome tissue sectioning, tissue dehydration with graded methanol solutions and careful critical point drying. Coupled with the helium ion system, fine details such as membrane texture and membranous nanoprojections on the glomerular podocytes were visualized, and pores within the filtration slit diaphragm could be seen in much greater detail than in previous scanning EM studies. In the collecting duct, the extensive and striking apical microplicae of the intercalated cells were imaged without the shrunken or distorted appearance that is typical with conventional sample processing and scanning electron microscopy. Membrane depressions visible on principal cells suggest possible endo- or exocytotic events, and central cilia on these cells were imaged with remarkable preservation and clarity. We also demonstrate the use of colloidal gold probes for highlighting specific cell-surface proteins and find that 15 nm gold labels are practical and easily distinguishable, indicating that external labels of various sizes can be used to detect multiple targets in the same tissue. We conclude that this technology represents a technical breakthrough in imaging the topographical ultrastructure of animal tissues. Its use in future studies should allow the study of fine cellular details and provide significant advances in our understanding of cell surface structures and membrane organization. PMID:23505418

Menadione metabolism to thiodione in hepatoblastoma by scanning electrochemical microscopy

PubMed Central

Mauzeroll, Janine; Bard, Allen J.; Owhadian, Omeed; Monks, Terrence J.

2004-01-01

The cytotoxicity of menadione on hepatocytes was studied by using the substrate generation/tip collection mode of scanning electrochemical microscopy by exposing the cells to menadione and detecting the menadione-S-glutathione conjugate (thiodione) that is formed during the cellular detoxication process and is exported from the cell by an ATP-dependent pump. This efflux was electrochemically detected and allowed scanning electrochemical microscopy monitoring and imaging of single cells and groups of highly confluent live cells. Based on a constant flux model, ≈6 × 106 molecules of thiodione per cell per second are exported from monolayer cultures of Hep G2 cells. PMID:15601769

Enhanced Axial Resolution of Wide-Field Two-Photon Excitation Microscopy by Line Scanning Using a Digital Micromirror Device.

PubMed

Park, Jong Kang; Rowlands, Christopher J; So, Peter T C

2017-01-01

Temporal focusing multiphoton microscopy is a technique for performing highly parallelized multiphoton microscopy while still maintaining depth discrimination. While the conventional wide-field configuration for temporal focusing suffers from sub-optimal axial resolution, line scanning temporal focusing, implemented here using a digital micromirror device (DMD), can provide substantial improvement. The DMD-based line scanning temporal focusing technique dynamically trades off the degree of parallelization, and hence imaging speed, for axial resolution, allowing performance parameters to be adapted to the experimental requirements. We demonstrate this new instrument in calibration specimens and in biological specimens, including a mouse kidney slice.

Enhanced Axial Resolution of Wide-Field Two-Photon Excitation Microscopy by Line Scanning Using a Digital Micromirror Device

PubMed Central

Park, Jong Kang; Rowlands, Christopher J.; So, Peter T. C.

2017-01-01

Temporal focusing multiphoton microscopy is a technique for performing highly parallelized multiphoton microscopy while still maintaining depth discrimination. While the conventional wide-field configuration for temporal focusing suffers from sub-optimal axial resolution, line scanning temporal focusing, implemented here using a digital micromirror device (DMD), can provide substantial improvement. The DMD-based line scanning temporal focusing technique dynamically trades off the degree of parallelization, and hence imaging speed, for axial resolution, allowing performance parameters to be adapted to the experimental requirements. We demonstrate this new instrument in calibration specimens and in biological specimens, including a mouse kidney slice. PMID:29387484

Note: Development of a wideband amplifier for cryogenic scanning tunneling microscopy.

PubMed

Zhang, Chao; Jeon, Hoyeon; Oh, Myungchul; Lee, Minjun; Kim, Sungmin; Yi, Sunwouk; Lee, Hanho; Zoh, Inhae; Yoo, Yongchan; Kuk, Young

2017-06-01

A wideband cryogenic amplifier has been developed for low temperature scanning tunneling microscopy. The amplifier consisting of a wideband complementary metal oxide semiconductor field effect transistors operational amplifier together with a feedback resistor of 100 kΩ and a capacitor is mounted within a 4 K Dewar. This amplifier has a wide bandwidth and is successfully applied to scanning tunneling microscopy applications at low temperatures down to ∼7 K. The quality of the designed amplifier is validated by high resolution imaging. More importantly, the amplifier has also proved to be capable of performing scanning tunneling spectroscopy measurements, showing the detection of the Shockley surface state of the Au(111) surface and the superconducting gap of Nb(110).

Note: Development of a wideband amplifier for cryogenic scanning tunneling microscopy

NASA Astrophysics Data System (ADS)

Zhang, Chao; Jeon, Hoyeon; Oh, Myungchul; Lee, Minjun; Kim, Sungmin; Yi, Sunwouk; Lee, Hanho; Zoh, Inhae; Yoo, Yongchan; Kuk, Young

2017-06-01

A wideband cryogenic amplifier has been developed for low temperature scanning tunneling microscopy. The amplifier consisting of a wideband complementary metal oxide semiconductor field effect transistors operational amplifier together with a feedback resistor of 100 kΩ and a capacitor is mounted within a 4 K Dewar. This amplifier has a wide bandwidth and is successfully applied to scanning tunneling microscopy applications at low temperatures down to ˜7 K. The quality of the designed amplifier is validated by high resolution imaging. More importantly, the amplifier has also proved to be capable of performing scanning tunneling spectroscopy measurements, showing the detection of the Shockley surface state of the Au(111) surface and the superconducting gap of Nb(110).

The Sounds of Nanoscience: Acoustic STM Analogues

ERIC Educational Resources Information Center

Euler, Manfred

2013-01-01

A hands-on model of scanning tunnelling microscopy (STM) is presented. It uses near-field imaging with sound and computer assisted visualization to create acoustic mappings of resonator arrangements. Due to the (partial) analogy of matter and sound waves the images closely resemble STM scans of atoms. Moreover, the method can be extended to build…

Ultrafast axial scanning for two-photon microscopy via a digital micromirror device and binary holography.

PubMed

Cheng, Jiyi; Gu, Chenglin; Zhang, Dapeng; Wang, Dien; Chen, Shih-Chi

2016-04-01

In this Letter, we present an ultrafast nonmechanical axial scanning method for two-photon excitation (TPE) microscopy based on binary holography using a digital micromirror device (DMD), achieving a scanning rate of 4.2 kHz, scanning range of ∼180  μm, and scanning resolution (minimum step size) of ∼270  nm. Axial scanning is achieved by projecting the femtosecond laser to a DMD programmed with binary holograms of spherical wavefronts of increasing/decreasing radii. To guide the scanner design, we have derived the parametric relationships between the DMD parameters (i.e., aperture and pixel size), and the axial scanning characteristics, including (1) maximum optical power, (2) minimum step size, and (3) scan range. To verify the results, the DMD scanner is integrated with a custom-built TPE microscope that operates at 60 frames per second. In the experiment, we scanned a pollen sample via both the DMD scanner and a precision z-stage. The results show the DMD scanner generates images of equal quality throughout the scanning range. The overall efficiency of the TPE system was measured to be ∼3%. With the high scanning rate, the DMD scanner may find important applications in random-access imaging or high-speed volumetric imaging that enables visualization of highly dynamic biological processes in 3D with submillisecond temporal resolution.

Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.

PubMed

Varga, Viktor Sebestyén; Bocsi, József; Sipos, Ferenc; Csendes, Gábor; Tulassay, Zsolt; Molnár, Béla

2004-07-01

Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. SFM is a valuable method for the evaluation of fluorescently labeled cells. Copyright 2004 Wiley-Liss, Inc.

Comparative study of image contrast in scanning electron microscope and helium ion microscope.

PubMed

O'Connell, R; Chen, Y; Zhang, H; Zhou, Y; Fox, D; Maguire, P; Wang, J J; Rodenburg, C

2017-12-01

Images of Ga + -implanted amorphous silicon layers in a 110 n-type silicon substrate have been collected by a range of detectors in a scanning electron microscope and a helium ion microscope. The effects of the implantation dose and imaging parameters (beam energy, dwell time, etc.) on the image contrast were investigated. We demonstrate a similar relationship for both the helium ion microscope Everhart-Thornley and scanning electron microscope Inlens detectors between the contrast of the images and the Ga + density and imaging parameters. These results also show that dynamic charging effects have a significant impact on the quantification of the helium ion microscope and scanning electron microscope contrast. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Cardiovascular Imaging Using Two-Photon Microscopy

PubMed Central

Scherschel, John A.; Rubart, Michael

2008-01-01

Two-photon excitation microscopy has become the standard technique for high resolution deep tissue and intravital imaging. It provides intrinsic three-dimensional resolution in combination with increased penetration depth compared to single-photon confocal microscopy. This article will describe the basic physical principles of two-photon excitation and will review its multiple applications to cardiovascular imaging, including second harmonic generation and fluorescence laser scanning microscopy. In particular, the capability and limitations of multiphoton microscopy to assess functional heterogeneity on a cellular scale deep within intact, Langendorff-perfused hearts are demonstrated. It will also discuss the use of two-photon excitation-induced release of caged compounds for the study of intracellular calcium signaling and intercellular dye transfer. PMID:18986603

Real-time high dynamic range laser scanning microscopy

NASA Astrophysics Data System (ADS)

Vinegoni, C.; Leon Swisher, C.; Fumene Feruglio, P.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R.

2016-04-01

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

Application of He ion microscopy for material analysis

NASA Astrophysics Data System (ADS)

Altmann, F.; Simon, M.; Klengel, R.

2009-05-01

Helium ion beam microscopy (HIM) is a new high resolution imaging technique. The use of Helium ions instead of electrons enables none destructive imaging combined with contrasts quite similar to that from Gallium ion beam imaging. The use of very low probe currents and the comfortable charge compensation using low energy electrons offer imaging of none conductive samples without conductive coating. An ongoing microelectronic sample with Gold/Aluminum interconnects and polymer electronic devices were chosen to evaluate HIM in comparison to scanning electron microscopy (SEM). The aim was to look for key applications of HIM in material analysis. Main focus was on complementary contrast mechanisms and imaging of none conductive samples.

Method for imaging liquid and dielectric materials with scanning polarization force microscopy

DOEpatents

Hu, J.; Ogletree, D.F.; Salmeron, M.; Xiao, X.

1999-03-09

The invention images dielectric polarization forces on surfaces induced by a charged scanning force microscope (SFM) probe tip. On insulators, the major contribution to the surface polarizability at low frequencies is from surface ions. The mobility of these ions depends strongly on the humidity. Using the inventive SFM, liquid films, droplets, and other weakly adsorbed materials have been imaged. 9 figs.

Apparatus for imaging liquid and dielectric materials with scanning polarization force microscopy

DOEpatents

Hu, J.; Ogletree, D.F.; Salmeron, M.; Xiao, X.

1998-04-28

The invention images dielectric polarization forces on surfaces induced by a charged scanning force microscope (SFM) probe tip. On insulators, the major contribution to the surface polarizability at low frequencies is from surface ions. The mobility of these ions depends strongly on the humidity. Using the inventive SFM, liquid films, droplets, and other weakly adsorbed materials have been imaged. 9 figs.

Microscopy refocusing and dark-field imaging by using a simple LED array.

PubMed

Zheng, Guoan; Kolner, Christopher; Yang, Changhuei

2011-10-15

The condenser is one of the main components in most transmitted light compound microscopes. In this Letter, we show that such a condenser can be replaced by a programmable LED array to achieve greater imaging flexibility and functionality. Without mechanically scanning the sample or changing the microscope setup, the proposed approach can be used for dark-field imaging, bright-field imaging, microscopy sectioning, and digital refocusing. Images of a starfish embryo were acquired by using such an approach for demonstration.

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

PubMed

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

2015-12-01

In this study, we introduce scanning electron microscopy (SEM) of semithin resin sections. In this technique, semithin sections were adhered on glass slides, stained with both uranyl acetate and lead citrate, and observed with a backscattered electron detector at a low accelerating voltage. As the specimens are stained in the same manner as conventional transmission electron microscopy (TEM), the contrast of SEM images of semithin sections was similar to TEM images of ultrathin sections. Using this technique, wide areas of semithin sections were also observed by SEM, without the obstruction of grids, which was inevitable for traditional TEM. This study also applied semithin section SEM to correlative light and electron microscopy. Correlative immunofluorescence microscopy and immune-SEM were performed in semithin sections of LR white resin-embedded specimens using a FluoroNanogold-labeled secondary antibody. Because LR white resin is hydrophilic and electron stable, this resin is suitable for immunostaining and SEM observation. Using correlative microscopy, the precise localization of the primary antibody was demonstrated by fluorescence microscopy and SEM. This method has great potential for studies examining the precise localization of molecules, including Golgi- and ER-associated proteins, in correlation with LM and SEM. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Imaging of biological macromolecules on mica in humid air by scanning electrochemical microscopy

PubMed Central

Fan, Fu-Ren F.; Bard, Allen J.

1999-01-01

Imaging of DNA, keyhole limpet hemocyanin, mouse monoclonal IgG, and glucose oxidase on a mica substrate has been accomplished by scanning electrochemical microscopy with a tungsten tip. The technique requires the use of a high relative humidity to form a thin film of water on the mica surface that allows electrochemical reactions to take place at the tip and produce a faradaic current (≈1 pA) that can be used to control tip position. The effect of relative humidity and surface pretreatment with buffer solutions on the ionic conductivity of a mica surface was investigated to find appropriate conditions for imaging. Resolution of the order of 1 nm was obtained. PMID:10588687

New Approach to Image Aerogels by Scanning Electron Microscopy

NASA Astrophysics Data System (ADS)

Solá, Francisco; Hurwitz, Frances; Yang, Jijing

2011-03-01

A new scanning electron microscopy (SEM) technique to image poor electrically conductive aerogels is presented. The process can be performed by non-expert SEM users. We showed that negative charging effects on aerogels can be minimized significantly by inserting dry nitrogen gas close to the region of interest. The process involves the local recombination of accumulated negative charges with positive ions generated from ionization processes. This new technique made possible the acquisition of images of aerogels with pores down to approximately 3nm in diameter using a positively biased Everhart-Thornley (E-T) detector. Well-founded concepts based on known models will also be presented with the aim to explain the results qualitatively.

High-resolution, high-throughput imaging with a multibeam scanning electron microscope.

PubMed

Eberle, A L; Mikula, S; Schalek, R; Lichtman, J; Knothe Tate, M L; Zeidler, D

2015-08-01

Electron-electron interactions and detector bandwidth limit the maximal imaging speed of single-beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from biological brain tissue to semiconductor wafers. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

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.

Measuring and imaging diffusion with multiple scan speed image correlation spectroscopy.

PubMed

Gröner, Nadine; Capoulade, Jérémie; Cremer, Christoph; Wachsmuth, Malte

2010-09-27

The intracellular mobility of biomolecules is determined by transport and diffusion as well as molecular interactions and is crucial for many processes in living cells. Methods of fluorescence microscopy like confocal laser scanning microscopy (CLSM) can be used to characterize the intracellular distribution of fluorescently labeled biomolecules. Fluorescence correlation spectroscopy (FCS) is used to describe diffusion, transport and photo-physical processes quantitatively. As an alternative to FCS, spatially resolved measurements of mobilities can be implemented using a CLSM by utilizing the spatio-temporal information inscribed into the image by the scan process, referred to as raster image correlation spectroscopy (RICS). Here we present and discuss an extended approach, multiple scan speed image correlation spectroscopy (msICS), which benefits from the advantages of RICS, i.e. the use of widely available instrumentation and the extraction of spatially resolved mobility information, without the need of a priori knowledge of diffusion properties. In addition, msICS covers a broad dynamic range, generates correlation data comparable to FCS measurements, and allows to derive two-dimensional maps of diffusion coefficients. We show the applicability of msICS to fluorophores in solution and to free EGFP in living cells.

High-speed 3D imaging of cellular activity in the brain using axially-extended beams and light sheets.

PubMed

Hillman, Elizabeth Mc; Voleti, Venkatakaushik; Patel, Kripa; Li, Wenze; Yu, Hang; Perez-Campos, Citlali; Benezra, Sam E; Bruno, Randy M; Galwaduge, Pubudu T

2018-06-01

As optical reporters and modulators of cellular activity have become increasingly sophisticated, the amount that can be learned about the brain via high-speed cellular imaging has increased dramatically. However, despite fervent innovation, point-scanning microscopy is facing a fundamental limit in achievable 3D imaging speeds and fields of view. A range of alternative approaches are emerging, some of which are moving away from point-scanning to use axially-extended beams or sheets of light, for example swept confocally aligned planar excitation (SCAPE) microscopy. These methods are proving effective for high-speed volumetric imaging of the nervous system of small organisms such as Drosophila (fruit fly) and D. Rerio (Zebrafish), and are showing promise for imaging activity in the living mammalian brain using both single and two-photon excitation. This article describes these approaches and presents a simple model that demonstrates key advantages of axially-extended illumination over point-scanning strategies for high-speed volumetric imaging, including longer integration times per voxel, improved photon efficiency and reduced photodamage. Copyright © 2018 Elsevier Ltd. All rights reserved.

En-face Flying Spot OCT/Ophthalmoscope

NASA Astrophysics Data System (ADS)

Rosen, Richard B.; Garcia, Patricia; Podoleanu, Adrian Gh.; Cucu, Radu; Dobre, George; Trifanov, Irina; van Velthoven, Mirjam E. J.; de Smet, Marc D.; Rogers, John A.; Hathaway, Mark; Pedro, Justin; Weitz, Rishard

This is a review of a technique for high-resolution imaging of the eye that allows multiple sample sectioning perspectives with different axial resolutions. The technique involves the flying spot approach employed in confocal scanning laser ophthalmoscopy which is extended to OCT imaging via time domain en face fast lateral scanning. The ability of imaging with multiple axial resolutions stimulated the development of the dual en face OCT-confocal imaging technology. Dual imaging also allows various other imaging combinations, such as OCT with confocal microscopy for imaging the eye anterior segment and OCT with fluorescence angiography imaging.

Cytology 3D structure formation based on optical microscopy images

NASA Astrophysics Data System (ADS)

Pronichev, A. N.; Polyakov, E. V.; Shabalova, I. P.; Djangirova, T. V.; Zaitsev, S. M.

2017-01-01

The article the article is devoted to optimization of the parameters of imaging of biological preparations in optical microscopy using a multispectral camera in visible range of electromagnetic radiation. A model for the image forming of virtual preparations was proposed. The optimum number of layers was determined for the object scan in depth and holistic perception of its switching according to the results of the experiment.

3D imaging of intrinsic crystalline defects in zinc oxide by spectrally resolved two-photon fluorescence microscopy

NASA Astrophysics Data System (ADS)

Al-Tabich, A.; Inami, W.; Kawata, Y.; Jablonski, R.; Worasawat, S.; Mimura, H.

2017-05-01

We present a method for three-dimensional intrinsic defect imaging in zinc oxide (ZnO) by spectrally resolved two-photon fluorescence microscopy, based on the previously presented method of observing a photoluminescence distribution in wide-gap semiconductor crystals [Noor et al., Appl. Phys. Lett. 92(16), 161106 (2008)]. A tightly focused light beam radiated by a titanium-sapphire laser is used to obtain a two-photon excitation of selected area of the ZnO sample. Photoluminescence intensity of a specific spectral range is then selected by optical band pass filters and measured by a photomultiplier tube. Reconstruction of the specimen image is done by scanning the volume of interest by a piezoelectric positioning stage and measuring the spectrally resolved photoluminescence intensity at each point. The method has been proved to be effective at locating intrinsic defects of the ZnO crystalline structure in the volume of the crystal. The method was compared with other defect imaging and 3D imaging techniques like scanning tunneling microscopy and confocal microscopy. In both cases, our method shows superior penetration abilities and, as the only method, allows location of the defects of the chosen type in 3D. In this paper, we present the results of oxygen vacancies and zinc antisites imaging in ZnO nanorods.

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

NASA Astrophysics Data System (ADS)

Upputuri, Paul Kumar; Pramanik, Manojit

2017-12-01

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

Advances in imaging and quantification of electrical properties at the nanoscale using Scanning Microwave Impedance Microscopy (sMIM)

NASA Astrophysics Data System (ADS)

Friedman, Stuart; Yang, Yongliang; Amster, Oskar

2015-03-01

Scanning Microwave Impedance Microscopy (sMIM) is a mode for Atomic Force Microscopy (AFM) enabling imaging of unique contrast mechanisms and measurement of local permittivity and conductivity at the 10's of nm length scale. Recent results will be presented illustrating high-resolution electrical features such as sub 15 nm Moire' patterns in Graphene, carbon nanotubes of various electrical states and ferro-electrics. In addition to imaging, the technique is suited to a variety of metrology applications where specific physical properties are determined quantitatively. We will present research activities on quantitative measurements using multiple techniques to determine dielectric constant (permittivity) and conductivity (e.g. dopant concentration) for a range of materials. Examples include bulk dielectrics, low-k dielectric thin films, capacitance standards and doped semiconductors. Funded in part by DOE SBIR DE-SC0009586.

Multiple microscopic approaches demonstrate linkage between chromoplast architecture and carotenoid composition in diverse Capsicum annuum fruit.

PubMed

Kilcrease, James; Collins, Aaron M; Richins, Richard D; Timlin, Jerilyn A; O'Connell, Mary A

2013-12-01

Increased accumulation of specific carotenoids in plastids through plant breeding or genetic engineering requires an understanding of the limitations that storage sites for these compounds may impose on that accumulation. Here, using Capsicum annuum L. fruit, we demonstrate directly the unique sub-organellar accumulation sites of specific carotenoids using live cell hyperspectral confocal Raman microscopy. Further, we show that chromoplasts from specific cultivars vary in shape and size, and these structural variations are associated with carotenoid compositional differences. Live-cell imaging utilizing laser scanning confocal (LSCM) and confocal Raman microscopy, as well as fixed tissue imaging by scanning and transmission electron microscopy (SEM and TEM), all demonstrated morphological differences with high concordance for the measurements across the multiple imaging modalities. These results reveal additional opportunities for genetic controls on fruit color and carotenoid-based phenotypes. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Imaging ion and molecular transport at subcellular resolution by secondary ion mass spectrometry

NASA Astrophysics Data System (ADS)

Chandra, Subhash; Morrison, George H.

1995-05-01

The transport of K+, Na+, and Ca2+ were imaged in individual cells with a Cameca IMS-3f ion microscope. Strict cryogenic frozen freeze-dry sample preparations were employed. Ion redistribution artifacts in conventional chemical preparations are discussed. Cryogenically prepared freeze-fractured freeze-dried cultured cells allowed the three-dimensional ion microscopic imaging of elements. As smaller structures in calcium images can be resolved with the 0.5 [mu]m spatial resolution, correlative techniques are needed to confirm their identity. The potentials of reflected light microscopy, scanning electron microscopy and laser scanning confocal microscopy are discussed for microfeature recognition in freeze-fractured freeze-dried cells. The feasibility of using frozen freeze-dried cells for imaging molecular transport at subcellular resolution was tested. Ion microscopy successfully imaged the transport of the isotopically tagged (13C, 15N) amino acid, -arginine. The labeled amino acid was imaged at mass 28 with a Cs+ primary ion beam as the 28(13C15N)- species. After a 4 h exposure of LLC-PK1 kidney cells to 4 mM labeled arginine, the amino acid was localized throughout the cell with a preferential incorporation into the nucleus and nucleolus. An example is also shown of the ion microscopic imaging of sodium borocaptate, an experimental therapeutic drug for brain tumors, in cryogenically prepared frozen freeze-dried Swiss 3T3 cells.

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

NASA Astrophysics Data System (ADS)

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

2018-05-01

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

Visualizing gold nanoparticle uptake in live cells with liquid scanning transmission electron microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2011-04-13

The intracellular uptake of 30 nm diameter gold nanoparticles (Au-NPs) was studied at the nanoscale in pristine eukaryotic cells. Live COS-7 cells were maintained in a microfluidic chamber and imaged using scanning transmission electron microscopy. A quantitative image analysis showed that Au-NPs bound to the membranes of vesicles, possibly lysosomes, and occupied 67% of the available surface area. The vesicles accumulated to form a micrometer-sized cluster after 24 h of incubation. Two clusters were analyzed and found to consist of 117 ± 9 and 164 ± 4 NP-filled vesicles.

Dynamic scan control in STEM: Spiral scans

DOE PAGES

Lupini, Andrew R.; Borisevich, Albina Y.; Kalinin, Sergei V.; ...

2016-06-13

Here, scanning transmission electron microscopy (STEM) has emerged as one of the foremost techniques to analyze materials at atomic resolution. However, two practical difficulties inherent to STEM imaging are: radiation damage imparted by the electron beam, which can potentially damage or otherwise modify the specimen and slow-scan image acquisition, which limits the ability to capture dynamic changes at high temporal resolution. Furthermore, due in part to scan flyback corrections, typical raster scan methods result in an uneven distribution of dose across the scanned area. A method to allow extremely fast scanning with a uniform residence time would enable imaging atmore » low electron doses, ameliorating radiation damage and at the same time permitting image acquisition at higher frame-rates while maintaining atomic resolution. The practical complication is that rastering the STEM probe at higher speeds causes significant image distortions. Non-square scan patterns provide a solution to this dilemma and can be tailored for low dose imaging conditions. Here, we develop a method for imaging with alternative scan patterns and investigate their performance at very high scan speeds. A general analysis for spiral scanning is presented here for the following spiral scan functions: Archimedean, Fermat, and constant linear velocity spirals, which were tested for STEM imaging. The quality of spiral scan STEM images is generally comparable with STEM images from conventional raster scans, and the dose uniformity can be improved.« less

Dynamic scan control in STEM: Spiral scans

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

Lupini, Andrew R.; Borisevich, Albina Y.; Kalinin, Sergei V.

Here, scanning transmission electron microscopy (STEM) has emerged as one of the foremost techniques to analyze materials at atomic resolution. However, two practical difficulties inherent to STEM imaging are: radiation damage imparted by the electron beam, which can potentially damage or otherwise modify the specimen and slow-scan image acquisition, which limits the ability to capture dynamic changes at high temporal resolution. Furthermore, due in part to scan flyback corrections, typical raster scan methods result in an uneven distribution of dose across the scanned area. A method to allow extremely fast scanning with a uniform residence time would enable imaging atmore » low electron doses, ameliorating radiation damage and at the same time permitting image acquisition at higher frame-rates while maintaining atomic resolution. The practical complication is that rastering the STEM probe at higher speeds causes significant image distortions. Non-square scan patterns provide a solution to this dilemma and can be tailored for low dose imaging conditions. Here, we develop a method for imaging with alternative scan patterns and investigate their performance at very high scan speeds. A general analysis for spiral scanning is presented here for the following spiral scan functions: Archimedean, Fermat, and constant linear velocity spirals, which were tested for STEM imaging. The quality of spiral scan STEM images is generally comparable with STEM images from conventional raster scans, and the dose uniformity can be improved.« less

Precision controlled atomic resolution scanning transmission electron microscopy using spiral scan pathways

NASA Astrophysics Data System (ADS)

Sang, Xiahan; Lupini, Andrew R.; Ding, Jilai; Kalinin, Sergei V.; Jesse, Stephen; Unocic, Raymond R.

2017-03-01

Atomic-resolution imaging in an aberration-corrected scanning transmission electron microscope (STEM) can enable direct correlation between atomic structure and materials functionality. The fast and precise control of the STEM probe is, however, challenging because the true beam location deviates from the assigned location depending on the properties of the deflectors. To reduce these deviations, i.e. image distortions, we use spiral scanning paths, allowing precise control of a sub-Å sized electron probe within an aberration-corrected STEM. Although spiral scanning avoids the sudden changes in the beam location (fly-back distortion) present in conventional raster scans, it is not distortion-free. “Archimedean” spirals, with a constant angular frequency within each scan, are used to determine the characteristic response at different frequencies. We then show that such characteristic functions can be used to correct image distortions present in more complicated constant linear velocity spirals, where the frequency varies within each scan. Through the combined application of constant linear velocity scanning and beam path corrections, spiral scan images are shown to exhibit less scan distortion than conventional raster scan images. The methodology presented here will be useful for in situ STEM imaging at higher temporal resolution and for imaging beam sensitive materials.

Precision controlled atomic resolution scanning transmission electron microscopy using spiral scan pathways.

PubMed

Sang, Xiahan; Lupini, Andrew R; Ding, Jilai; Kalinin, Sergei V; Jesse, Stephen; Unocic, Raymond R

2017-03-08

Atomic-resolution imaging in an aberration-corrected scanning transmission electron microscope (STEM) can enable direct correlation between atomic structure and materials functionality. The fast and precise control of the STEM probe is, however, challenging because the true beam location deviates from the assigned location depending on the properties of the deflectors. To reduce these deviations, i.e. image distortions, we use spiral scanning paths, allowing precise control of a sub-Å sized electron probe within an aberration-corrected STEM. Although spiral scanning avoids the sudden changes in the beam location (fly-back distortion) present in conventional raster scans, it is not distortion-free. "Archimedean" spirals, with a constant angular frequency within each scan, are used to determine the characteristic response at different frequencies. We then show that such characteristic functions can be used to correct image distortions present in more complicated constant linear velocity spirals, where the frequency varies within each scan. Through the combined application of constant linear velocity scanning and beam path corrections, spiral scan images are shown to exhibit less scan distortion than conventional raster scan images. The methodology presented here will be useful for in situ STEM imaging at higher temporal resolution and for imaging beam sensitive materials.

Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry

DOE PAGES

Ophus, Colin; Ciston, Jim; Pierce, Jordan; ...

2016-02-29

The ability to image light elements in soft matter at atomic resolution enables unprecedented insight into the structure and properties of molecular heterostructures and beam-sensitive nanomaterials. In this study, we introduce a scanning transmission electron microscopy technique combining a pre-specimen phase plate designed to produce a probe with structured phase with a high-speed direct electron detector to generate nearly linear contrast images with high efficiency. We demonstrate this method by using both experiment and simulation to simultaneously image the atomic-scale structure of weakly scattering amorphous carbon and strongly scattering gold nanoparticles. Our method demonstrates strong contrast for both materials, makingmore » it a promising candidate for structural determination of heterogeneous soft/hard matter samples even at low electron doses comparable to traditional phase-contrast transmission electron microscopy. Ultimately, simulated images demonstrate the extension of this technique to the challenging problem of structural determination of biological material at the surface of inorganic crystals.« less

Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry.

PubMed

Ophus, Colin; Ciston, Jim; Pierce, Jordan; Harvey, Tyler R; Chess, Jordan; McMorran, Benjamin J; Czarnik, Cory; Rose, Harald H; Ercius, Peter

2016-02-29

The ability to image light elements in soft matter at atomic resolution enables unprecedented insight into the structure and properties of molecular heterostructures and beam-sensitive nanomaterials. In this study, we introduce a scanning transmission electron microscopy technique combining a pre-specimen phase plate designed to produce a probe with structured phase with a high-speed direct electron detector to generate nearly linear contrast images with high efficiency. We demonstrate this method by using both experiment and simulation to simultaneously image the atomic-scale structure of weakly scattering amorphous carbon and strongly scattering gold nanoparticles. Our method demonstrates strong contrast for both materials, making it a promising candidate for structural determination of heterogeneous soft/hard matter samples even at low electron doses comparable to traditional phase-contrast transmission electron microscopy. Simulated images demonstrate the extension of this technique to the challenging problem of structural determination of biological material at the surface of inorganic crystals.

Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry

PubMed Central

Ophus, Colin; Ciston, Jim; Pierce, Jordan; Harvey, Tyler R.; Chess, Jordan; McMorran, Benjamin J.; Czarnik, Cory; Rose, Harald H.; Ercius, Peter

2016-01-01

The ability to image light elements in soft matter at atomic resolution enables unprecedented insight into the structure and properties of molecular heterostructures and beam-sensitive nanomaterials. In this study, we introduce a scanning transmission electron microscopy technique combining a pre-specimen phase plate designed to produce a probe with structured phase with a high-speed direct electron detector to generate nearly linear contrast images with high efficiency. We demonstrate this method by using both experiment and simulation to simultaneously image the atomic-scale structure of weakly scattering amorphous carbon and strongly scattering gold nanoparticles. Our method demonstrates strong contrast for both materials, making it a promising candidate for structural determination of heterogeneous soft/hard matter samples even at low electron doses comparable to traditional phase-contrast transmission electron microscopy. Simulated images demonstrate the extension of this technique to the challenging problem of structural determination of biological material at the surface of inorganic crystals. PMID:26923483

3D imaging of cement-based materials at submicron resolution by combining laser scanning confocal microscopy with serial sectioning.

PubMed

Yio, M H N; Mac, M J; Wong, H S; Buenfeld, N R

2015-05-01

In this paper, we present a new method to reconstruct large volumes of nontransparent porous materials at submicron resolution. The proposed method combines fluorescence laser scanning confocal microscopy with serial sectioning to produce a series of overlapping confocal z-stacks, which are then aligned and stitched based on phase correlation. The method can be extended in the XY plane to further increase the overall image volume. Resolution of the reconstructed image volume does not degrade with increase in sample size. We have used the method to image cementitious materials, hardened cement paste and concrete and the results obtained show that the method is reliable. Possible applications of the method such as three-dimensional characterization of the pores and microcracks in hardened concrete, three-dimensional particle shape characterization of cementitious materials and three-dimensional characterization of other porous materials such as rocks and bioceramics are discussed. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

A Versatile High-Vacuum Cryo-transfer System for Cryo-microscopy and Analytics

PubMed Central

Tacke, Sebastian; Krzyzanek, Vladislav; Nüsse, Harald; Wepf, Roger Albert; Klingauf, Jürgen; Reichelt, Rudolf

2016-01-01

Cryogenic microscopy methods have gained increasing popularity, as they offer an unaltered view on the architecture of biological specimens. As a prerequisite, samples must be handled under cryogenic conditions below their recrystallization temperature, and contamination during sample transfer and handling must be prevented. We present a high-vacuum cryo-transfer system that streamlines the entire handling of frozen-hydrated samples from the vitrification process to low temperature imaging for scanning transmission electron microscopy and transmission electron microscopy. A template for cryo-electron microscopy and multimodal cryo-imaging approaches with numerous sample transfer steps is presented. PMID:26910419

Two-Photon Fluorescence Microscopy Developed for Microgravity Fluid Physics

NASA Technical Reports Server (NTRS)

Fischer, David G.; Zimmerli, Gregory A.; Asipauskas, Marius

2004-01-01

Recent research efforts within the Microgravity Fluid Physics Branch of the NASA Glenn Research Center have necessitated the development of a microscope capable of high-resolution, three-dimensional imaging of intracellular structure and tissue morphology. Standard optical microscopy works well for thin samples, but it does not allow the imaging of thick samples because of severe degradation caused by out-of-focus object structure. Confocal microscopy, which is a laser-based scanning microscopy, provides improved three-dimensional imaging and true optical sectioning by excluding the out-of-focus light. However, in confocal microscopy, out-of-focus object structure is still illuminated by the incoming beam, which can lead to substantial photo-bleaching. In addition, confocal microscopy is plagued by limited penetration depth, signal loss due to the presence of a confocal pinhole, and the possibility of live-cell damage. Two-photon microscopy is a novel form of laser-based scanning microscopy that allows three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon microscopy, it utilizes the nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption because of the nonlinear (i.e., quadratic) electric field dependence, so an ultrafast pulsed laser source must typically be employed. On the other hand, this stringent energy density requirement effectively localizes fluorophore excitation to the focal volume. Consequently, two-photon microscopy provides optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction in photo-damage because of the longer excitation wavelength, a reduction in background fluorescence, and a 4 increase in penetration depth over confocal methods because of the reduction in Rayleigh scattering.

Imaging of subunit complexes of thermophilic bacterium H(+)-ATPase with scanning tunneling microscopy.

PubMed

Masai, J; Shibata, T; Kagawa, Y; Kondo, S

1992-07-01

Using a scanning tunneling microscope (STM), we observed reconstructed subunit complexes of H(+)-ATPase of a thermophilic bacterium. The measurement was carried out in air without conductive coating on the samples deposited on a highly oriented pyrolytic graphite (HOPG). The F1 subunit complex of the H(+)-ATPase, and an H(+)-ATPase whose F0 portion was embedded into liposomes prepared from soybean lecithin were imaged. Overall structural images of the subunit complex F1 were obtained: the structural dimensions of the STM images are in agreement with those deduced from conventional methods such as an transmission electron microscopy (TEM) and small-angle X-ray scattering (SAX) experimentation. Regarding the STM imaging of these samples, we discuss the advantages and disadvantages of the STM over those of conventional methods such as a TEM and SAX.

Wide-field two-photon microscopy with temporal focusing and HiLo background rejection

NASA Astrophysics Data System (ADS)

Yew, Elijah Y. S.; Choi, Heejin; Kim, Daekeun; So, Peter T. C.

2011-03-01

Scanningless depth-resolved microscopy is achieved through spatial-temporal focusing and has been demonstrated previously. The advantage of this method is that a large area may be imaged without scanning resulting in higher throughput of the imaging system. Because it is a widefield technique, the optical sectioning effect is considerably poorer than with conventional spatial focusing two-photon microscopy. Here we propose wide-field two-photon microscopy based on spatio-temporal focusing and employing background rejection based on the HiLo microscope principle. We demonstrate the effects of applying HiLo microscopy to widefield temporally focused two-photon microscopy.

Tip/tilt-compensated through-focus scanning optical microscopy

NASA Astrophysics Data System (ADS)

Lee, Jun Ho; Park, Jun Hyung; Jeong, Dohwan; Shin, Eun Ji; Park, Chris

2016-11-01

Through-Focus Optical Microscopy (TSOM), with nanometer scale lateral and vertical sensitivity matching those of scanning electron microscopy, has been demonstrated to be utilized for 3D inspection and metrology. There have been sensitivity and instability issues in acquiring through-focus images because TSOM 3D information is indirectly extracted by differentiating a target TSOM image from reference TSOM images. This paper first reports on the optical axis instability that occurs during the scanning process of TSOM when implemented in an existing patterned wafer inspection tool by moving the wafer plane; this is followed by quantitative confirmation of the optical/mechanical instability using a new TSOM tool on an optical bench with a Shack-Hartmann wavefront sensor and a tip/tilt sensor. Then, this paper proposes two tip/tilt compensated TSOM optical acquisition methods that can be applied with adaptive optics. The first method simply adopts a tip/tilt mirror with a quad cell in a simple closed loop, while the second method adopts a highorder deformable mirror with a Shack-Hartmann sensor. The second method is able to correct high-order residual aberrations as well as to perform through-focus scanning without z-axis movement, while the first method is easier to implement in pre-existing wafer inspection systems with only minor modification.

Imaging a Large Sample with Selective Plane Illumination Microscopy Based on Multiple Fluorescent Microsphere Tracking

NASA Astrophysics Data System (ADS)

Ryu, Inkeon; Kim, Daekeun

2018-04-01

A typical selective plane illumination microscopy (SPIM) image size is basically limited by the field of view, which is a characteristic of the objective lens. If an image larger than the imaging area of the sample is to be obtained, image stitching, which combines step-scanned images into a single panoramic image, is required. However, accurately registering the step-scanned images is very difficult because the SPIM system uses a customized sample mount where uncertainties for the translational and the rotational motions exist. In this paper, an image registration technique based on multiple fluorescent microsphere tracking is proposed in the view of quantifying the constellations and measuring the distances between at least two fluorescent microspheres embedded in the sample. Image stitching results are demonstrated for optically cleared large tissue with various staining methods. Compensation for the effect of the sample rotation that occurs during the translational motion in the sample mount is also discussed.

Automated motion artifact removal for intravital microscopy, without a priori information.

PubMed

Lee, Sungon; Vinegoni, Claudio; Sebas, Matthew; Weissleder, Ralph

2014-03-28

Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber.

Automated motion artifact removal for intravital microscopy, without a priori information

PubMed Central

Lee, Sungon; Vinegoni, Claudio; Sebas, Matthew; Weissleder, Ralph

2014-01-01

Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber. PMID:24676021

Reassignment of scattered emission photons in multifocal multiphoton microscopy.

PubMed

Cha, Jae Won; Singh, Vijay Raj; Kim, Ki Hean; Subramanian, Jaichandar; Peng, Qiwen; Yu, Hanry; Nedivi, Elly; So, Peter T C

2014-06-05

Multifocal multiphoton microscopy (MMM) achieves fast imaging by simultaneously scanning multiple foci across different regions of specimen. The use of imaging detectors in MMM, such as CCD or CMOS, results in degradation of image signal-to-noise-ratio (SNR) due to the scattering of emitted photons. SNR can be partly recovered using multianode photomultiplier tubes (MAPMT). In this design, however, emission photons scattered to neighbor anodes are encoded by the foci scan location resulting in ghost images. The crosstalk between different anodes is currently measured a priori, which is cumbersome as it depends specimen properties. Here, we present the photon reassignment method for MMM, established based on the maximum likelihood (ML) estimation, for quantification of crosstalk between the anodes of MAPMT without a priori measurement. The method provides the reassignment of the photons generated by the ghost images to the original spatial location thus increases the SNR of the final reconstructed image.

Atomic bonding effects in annular dark field scanning transmission electron microscopy. I. Computational predictions

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

Odlyzko, Michael L.; Mkhoyan, K. Andre, E-mail: mkhoyan@umn.edu; Himmetoglu, Burak

2016-07-15

Annular dark field scanning transmission electron microscopy (ADF-STEM) image simulations were performed for zone-axis-oriented light-element single crystals, using a multislice method adapted to include charge redistribution due to chemical bonding. Examination of these image simulations alongside calculations of the propagation of the focused electron probe reveal that the evolution of the probe intensity with thickness exhibits significant sensitivity to interatomic charge transfer, accounting for observed thickness-dependent bonding sensitivity of contrast in all ADF-STEM imaging conditions. Because changes in image contrast relative to conventional neutral atom simulations scale directly with the net interatomic charge transfer, the strongest effects are seen inmore » crystals with highly polar bonding, while no effects are seen for nonpolar bonding. Although the bonding dependence of ADF-STEM image contrast varies with detector geometry, imaging parameters, and material temperature, these simulations predict the bonding effects to be experimentally measureable.« less

Bright-field scanning confocal electron microscopy using a double aberration-corrected transmission electron microscope.

PubMed

Wang, Peng; Behan, Gavin; Kirkland, Angus I; Nellist, Peter D; Cosgriff, Eireann C; D'Alfonso, Adrian J; Morgan, Andrew J; Allen, Leslie J; Hashimoto, Ayako; Takeguchi, Masaki; Mitsuishi, Kazutaka; Shimojo, Masayuki

2011-06-01

Scanning confocal electron microscopy (SCEM) offers a mechanism for three-dimensional imaging of materials, which makes use of the reduced depth of field in an aberration-corrected transmission electron microscope. The simplest configuration of SCEM is the bright-field mode. In this paper we present experimental data and simulations showing the form of bright-field SCEM images. We show that the depth dependence of the three-dimensional image can be explained in terms of two-dimensional images formed in the detector plane. For a crystalline sample, this so-called probe image is shown to be similar to a conventional diffraction pattern. Experimental results and simulations show how the diffracted probes in this image are elongated in thicker crystals and the use of this elongation to estimate sample thickness is explored. Copyright © 2010 Elsevier B.V. All rights reserved.

EDITORIAL: Scanning probe microscopy: a visionary development Scanning probe microscopy: a visionary development

NASA Astrophysics Data System (ADS)

Demming, Anna

2013-07-01

The development of scanning probe microscopy repositioned modern physics. When Rohrer and Binnig first used electronic tunnelling effects to image atoms and quantum states they did more than pin down theoretical hypotheses to real-world observables; the scanning tunnelling microscope fed imaginations, prompting researchers to consider new directions and possibilities [1]. As Rohrer once commented, 'We could show that you can easily manipulate or position something small in space with an accuracy of 10 pm.... When you can do that, you simply have ideas of what you can do' [2]. The development heralded a cavalry of scanning probe techniques—such as atomic force microscopy (AFM) [3-5], scanning near-field optical microscopy (SNOM) [6-8] and Kelvin probe force microscopy (KPFM) [9, 10]—that still continue to bring nanomaterials and nanoscale phenomena into fresh focus. Not long after the development of scanning tunnelling microscopy, Binnig, Quate and Gerber collaborating in California in the US published work on a new type of microscope also capable of atomic level resolution [3]. The original concept behind scanning tunnelling microscopy uses electrical conductance, which places substantial limitations on the systems that it can image. Binnig, Quate and Gerber developed the AFM to 'feel' the topology of surfaces like the needle of an old fashioned vinyl player. In this way insulators could be imaged as well. The development of a force modulation mode AFM extended the tool's reach to soft materials making images of biological samples accessible with the technique [4]. There have now been a number of demonstrations of image capture at rates that allow dynamics at the nanoscale to be tracked in real time, opening further possibilities in applications of the AFM as described in a recent review by Toshio Ando at Kanazawa University [5]. Researchers also found a way to retrieve optical information at 'super-resolution' [6, 7]. Optical microscopy provides spectral details that harbour a wealth of additional information about the sample and its environment, like switching from black and white to technicolour. With the invention of SNOM these details were no longer restricted by the diffraction limit to a resolution of half the wavelength of the incident light. The principle behind SNOM remains very similar to STM but instead of measuring an electronic current, information is captured from the non-propagating optical near field, where the diffraction limit does not apply. SNOM continues to be an invaluable imaging technique as demonstrated recently by researchers in Spain and Korea, who used it to measure near-infrared-to-visible upconversion and cathodoluminescence emission properties of Ln3+ in nanocrystalline Ln-doped Lu2O3 materials with 1D morphology [8]. Their work holds promise for controlled incorporation of such optically active nanostructures in future photonic structures and applications. The cantilever-probe system provides a number of highly sensitive interactions that can be exploited to extract details of a sample system. The potential offset between the probe and surface manifests itself in a force and this too has been used in KPFM [9]. The finite tip size has a profound effect on the measured image in scanning probe-microscopes in general. In KPFM, as Rosenwaks and colleagues in Israel, US and Germany point out in this issue [10] the influence of the tip and cantilever on measurements is particularly significant because of the long range nature of the electrostatic forces involved. Measurements at any one point provide a weighted average of the contact potential difference of the sample and to obtain a quantitative image this averaging must be taken into account. Rosenwaks and colleagues tackle this challenge in the work reported in this issue, presenting an algorithm for reconstructing a sample surface potential from its KPFM image. Their study also reveals that the averaging effects are far more significant for amplitude modulated KPFM measurements compared with the frequency modulated mode. Rohrer and Binnig shared the Nobel Prize for Physics 'for their design of the scanning tunnelling microscope' [11]. They are widely recognized among the founding fathers of nanoscience. In an interview in 2005 Rohrer once commented on the benefits of changing fields even if it leaves you feeling a little 'lost and lonely' at first. In fact he attributed his ability to contribute his Nobel Prize winning work to science at a comparatively senior age to the fact that he had changed fields. 'You cannot be the star from the beginning, but I think what is important is that you might bring in a different way of thinking. You have a certain lightness to approach something that is the expert opinion' [2]. In nanotechnology where such a formidable range of disciplines seem to feed into the research such words may be particularly encouraging. Rohrer passed away on 16 May 2013, but the awesome legacy of his life's work continues. With the scanning tunnelling microscope the lofty eccentricities of quantum mechanical theory literally came into view, quite an inspiration. References [1] Binning G, Rohrer H, Gerber Ch and Weibel E 1982 Surface studies by scanning tunneling microscopy Phys. Rev. Lett. 49 57-61 [2] Weiss P S 2007 A conversation with Dr. Heinrich Rohrer: STM Co-inventor and one of the founding fathers of nanoscience ACS Nano 1 3-5 [3] Binnig G, Quate C F and Gerber Ch 1986 Atomic force microscope Phys. Rev. Lett. 56 930-3 [4] Maivald P, Butt H J, Gould S A C, Prater C B, Drake B, Gurley J A, Elings V B and Hansma P K 1991 Using force modulation to image surface elasticities with the atomic force microscope Nanotechnology 2 103-6 [5] Ando T 2012 High-speed atomic force microscopy coming of age Nanotechnology 23 062001 [6] Betzig E, Isaacson M, Barshatzky H, Lewis A and Lin K 1988 Super-resolution imaging with near-field scanning optical microscopy (NSOM) Ultramicroscopy 25 155-63 [7] Thio T, Lezec H J, Ebbesen T W, Pellerin K M, Lewen G D, Nahata A and Linke R A 2002 Giant optical transmission of sub-wavelength apertures: physics and applications Nanotechnology 13 429-32 [8] Barrera E W, Pujol M C, Díaz F, Choi S B, Rotermund F, Park K H, Jeong M S and Cascales C 2011 Emission properties of hydrothermal Yb3+, Er3+ and Yb3+, Tm3+-codoped Lu2O3 nanorods: upconversion, cathodoluminescence and assessment of waveguide behaviour Nanotechnology 22 075205 [9] Nonnenmacher M, O'Boyle M P and Wickramasinghe H K 1991 Kelvin probe force microscopy Appl. Phys. Lett. 58 2921-3 [10] Cohen G, Halpern E, Nanayakkara S U, Luther J M, Held C, Bennewitz R, Boag A and Rosenwaks Y 2013 Reconstruction of surface potential from Kelvin probe force microscopy images Nanotechnology 24 295702 [11] 1986 The Nobel Prize in Physics www.nobelprize.org/nobel prizes/physics/laureates/1986/ index.html

Electron microscopy localization and characterization of functionalized composite organic-inorganic SERS nanoparticles on leukemia cells.

PubMed

Koh, Ai Leen; Shachaf, Catherine M; Elchuri, Sailaja; Nolan, Garry P; Sinclair, Robert

2008-12-01

We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2013-01-01

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

In vivo microscopy of the mouse brain using multiphoton laser scanning techniques

NASA Astrophysics Data System (ADS)

Yoder, Elizabeth J.

2002-06-01

The use of multiphoton microscopy for imaging mouse brain in vivo offers several advantages and poses several challenges. This tutorial begins by briefly comparing multiphoton microscopy with other imaging modalities used to visualize the brain and its activity. Next, an overview of the techniques for introducing fluorescence into whole animals to generate contrast for in vivo microscopy using two-photon excitation is presented. Two different schemes of surgically preparing mice for brain imaging with multiphoton microscopy are reviewed. Then, several issues and problems with in vivo microscopy - including motion artifact, respiratory and cardiac rhythms, maintenance of animal health, anesthesia, and the use of fiducial markers - are discussed. Finally, examples of how these techniques have been applied to visualize the cerebral vasculature and its response to hypercapnic stimulation are provided.

High-speed atomic force microscopy imaging of live mammalian cells

PubMed Central

Shibata, Mikihiro; Watanabe, Hiroki; Uchihashi, Takayuki; Ando, Toshio; Yasuda, Ryohei

2017-01-01

Direct imaging of morphological dynamics of live mammalian cells with nanometer resolution under physiological conditions is highly expected, but yet challenging. High-speed atomic force microscopy (HS-AFM) is a unique technique for capturing biomolecules at work under near physiological conditions. However, application of HS-AFM for imaging of live mammalian cells was hard to be accomplished because of collision between a huge mammalian cell and a cantilever during AFM scanning. Here, we review our recent improvements of HS-AFM for imaging of activities of live mammalian cells without significant damage to the cell. The improvement of an extremely long (~3 μm) AFM tip attached to a cantilever enables us to reduce severe damage to soft mammalian cells. In addition, a combination of HS-AFM with simple fluorescence microscopy allows us to quickly locate the cell in the AFM scanning area. After these improvements, we demonstrate that developed HS-AFM for live mammalian cells is possible to image morphogenesis of filopodia, membrane ruffles, pits open-close formations, and endocytosis in COS-7, HeLa cells as well as hippocampal neurons. PMID:28900590

High resolution surface plasmon microscopy for cell imaging

NASA Astrophysics Data System (ADS)

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

2010-04-01

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

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.

Real-time high dynamic range laser scanning microscopy

PubMed Central

Vinegoni, C.; Leon Swisher, C.; Fumene Feruglio, P.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R.

2016-01-01

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging. PMID:27032979

Challenges of microtome‐based serial block‐face scanning electron microscopy in neuroscience

PubMed Central

WANNER, A. A.; KIRSCHMANN, M. A.

2015-01-01

Summary Serial block‐face scanning electron microscopy (SBEM) is becoming increasingly popular for a wide range of applications in many disciplines from biology to material sciences. This review focuses on applications for circuit reconstruction in neuroscience, which is one of the major driving forces advancing SBEM. Neuronal circuit reconstruction poses exceptional challenges to volume EM in terms of resolution, field of view, acquisition time and sample preparation. Mapping the connections between neurons in the brain is crucial for understanding information flow and information processing in the brain. However, information on the connectivity between hundreds or even thousands of neurons densely packed in neuronal microcircuits is still largely missing. Volume EM techniques such as serial section TEM, automated tape‐collecting ultramicrotome, focused ion‐beam scanning electron microscopy and SBEM (microtome serial block‐face scanning electron microscopy) are the techniques that provide sufficient resolution to resolve ultrastructural details such as synapses and provides sufficient field of view for dense reconstruction of neuronal circuits. While volume EM techniques are advancing, they are generating large data sets on the terabyte scale that require new image processing workflows and analysis tools. In this review, we present the recent advances in SBEM for circuit reconstruction in neuroscience and an overview of existing image processing and analysis pipelines. PMID:25907464

Suitability of holographic beam scanning in high resolution applications

NASA Astrophysics Data System (ADS)

Kalita, Ranjan; Goutam Buddha, S. S.; Boruah, Bosanta R.

2018-02-01

The high resolution applications of a laser scanning imaging system very much demand the accurate positioning of the illumination beam. The galvanometer scanner based beam scanning imaging systems, on the other hand, suffer from both short term and long term beam instability issues. Fortunately Computer generated holography based beam scanning offers extremely accurate beam steering, which can be very useful for imaging in high-resolution applications in confocal microscopy. The holographic beam scanning can be achieved by writing a sequence of holograms onto a spatial light modulator and utilizing one of the diffracted orders as the illumination beam. This paper highlights relative advantages of such a holographic beam scanning based confocal system and presents some of preliminary experimental results.

Low-temperature and conventional scanning electron microscopy of human urothelial neoplasms.

PubMed

Hopkins, D M; Morris, J A; Oates, K; Huddart, H; Staff, W G

1989-05-01

The appearance of neoplastic human urothelium viewed by low-temperature scanning electron microscopy (LTSEM) and conventional scanning electron microscopy (CSEM) was compared. Fixed, dehydrated neoplastic cells viewed by CSEM had well-defined, often raised cell junctions; no intercellular gaps; and varying degrees of pleomorphic surface microvilli. The frozen hydrated material viewed by LTSEM, however, was quite different. The cells had a flat or dimpled surface, but no microvilli. There were labyrinthine lateral processes which interdigitated with those of adjacent cells and outlined large intercellular gaps. The process of fixation and dehydration will inevitably distort cell contours and on theoretical grounds, the images of frozen hydrated material should more closely resemble the in vivo appearance.

Nanoscale live cell imaging using hopping probe ion conductance microscopy

PubMed Central

Novak, Pavel; Li, Chao; Shevchuk, Andrew I.; Stepanyan, Ruben; Caldwell, Matthew; Hughes, Simon; Smart, Trevor G.; Gorelik, Julia; Ostanin, Victor P.; Lab, Max J.; Moss, Guy W. J.; Frolenkov, Gregory I.; Klenerman, David; Korchev, Yuri E.

2009-01-01

We describe a major advance in scanning ion conductance microscopy: a new hopping mode that allows non-contact imaging of the complex surfaces of live cells with resolution better than 20 nm. The effectiveness of this novel technique was demonstrated by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allows studying nanoscale phenomena on the surface of live cells under physiological conditions. PMID:19252505

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).

Secretory glands and microvascular systems imaged in aqueous solution by atmospheric scanning electron microscopy (ASEM).

PubMed

Yamazawa, Toshiko; Nakamura, Naotoshi; Sato, Mari; Sato, Chikara

2016-12-01

Exocrine glands, e.g., salivary and pancreatic glands, play an important role in digestive enzyme secretion, while endocrine glands, e.g., pancreatic islets, secrete hormones that regulate blood glucose levels. The dysfunction of these secretory organs immediately leads to various diseases, such as diabetes or Sjögren's syndrome, by poorly understood mechanisms. Gland-related diseases have been studied by optical microscopy (OM), and at higher resolution by transmission electron microscopy (TEM) of Epon embedded samples, which necessitates hydrophobic sample pretreatment. Here, we report the direct observation of tissue in aqueous solution by atmospheric scanning electron microscopy (ASEM). Salivary glands, lacrimal glands, and pancreas were fixed, sectioned into slabs, stained with phosphotungstic acid (PTA), and inspected in radical scavenger d-glucose solution from below by an inverted scanning electron microscopy (SEM), guided by optical microscopy from above to target the tissue substructures. A 2- to 3-µm specimen thickness was visualized by the SEM. In secretory cells, cytoplasmic vesicles and other organelles were clearly imaged at high resolution, and the former could be classified according to the degree of PTA staining. In islets of Langerhans, the microvascular system used as an outlet by the secretory cells was also clearly observed. Microvascular system is also critically involved in the onset of diabetic complications and was clearly visible in subcutaneous tissue imaged by ASEM. The results suggest the use of in-solution ASEM for histology and to study vesicle secretion systems. Further, the high-throughput of ASEM makes it a potential tool for the diagnosis of exocrine and endocrine-related diseases. © 2016 Wiley Periodicals, Inc.

Correlative Fluorescence and Electron Microscopy in 3D-Scanning Electron Microscope Perspective.

PubMed

Franks, Jonathan; Wallace, Callen T; Shibata, Masateru; Suga, Mitsuo; Erdman, Natasha; Stolz, Donna B; Watkins, Simon C

2017-04-03

The ability to correlate fluorescence microscopy (FM) and electron microscopy (EM) data obtained on biological (cell and tissue) specimens is essential to bridge the resolution gap between the data obtained by these different imaging techniques. In the past such correlations were limited to either EM navigation in two dimensions to the locations previously highlighted by fluorescence markers, or subsequent high-resolution acquisition of tomographic information using a TEM. We present a novel approach whereby a sample previously investigated by FM is embedded and subjected to sequential mechanical polishing and backscatter imaging by scanning electron microscope. The resulting three dimensional EM tomogram of the sample can be directly correlated to the FM data. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

A digital atlas of breast histopathology: an application of web based virtual microscopy

PubMed Central

Lundin, M; Lundin, J; Helin, H; Isola, J

2004-01-01

Aims: To develop an educationally useful atlas of breast histopathology, using advanced web based virtual microscopy technology. Methods: By using a robotic microscope and software adopted and modified from the aerial and satellite imaging industry, a virtual microscopy system was developed that allows fully automated slide scanning and image distribution via the internet. More than 150 slides were scanned at high resolution with an oil immersion ×40 objective (numerical aperture, 1.3) and archived on an image server residing in a high speed university network. Results: A publicly available website was constructed, http://www.webmicroscope.net/breastatlas, which features a comprehensive virtual slide atlas of breast histopathology according to the World Health Organisation 2003 classification. Users can view any part of an entire specimen at any magnification within a standard web browser. The virtual slides are supplemented with concise textual descriptions, but can also be viewed without diagnostic information for self assessment of histopathology skills. Conclusions: Using the technology described here, it is feasible to develop clinically and educationally useful virtual microscopy applications. Web based virtual microscopy will probably become widely used at all levels in pathology teaching. PMID:15563669

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

Confocal laser scanning microscopy of porcine skin: implications for human wound healing studies

PubMed Central

VARDAXIS, N. J.; BRANS, T. A.; BOON, M. E.; KREIS, R. W.; MARRES, L. M.

1997-01-01

The structure of porcine skin as examined by light microscopy is reviewed and its similarities to and differences from human skin are highlighted. Special imaging techniques and staining procedures are described and their use in gathering morphological information in porcine skin is discussed. Confocal laser scanning microscopy (CLSM) was employed to examine the structure of porcine skin and the findings are presented as an adjunct to the information already available in the literature. It is concluded that CLSM provides valuable additional morphological information to material examined by conventional microscopy and is useful for wound healing studies in the porcine model. PMID:9183682

Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues

PubMed Central

Benninger, Richard K.P.; Piston, David W.

2013-01-01

Two-photon excitation microscopy is an alternative to confocal microscopy that provides advantages for three-dimensional and deep tissue imaging. This unit will describe the basic physical principles behind two-photon excitation and discuss the advantages and limitations of its use in laser-scanning microscopy. The principal advantages of two-photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate highly localized photochemistry in thick samples. Practical considerations for the application of two-photon microscopy will then be discussed, including recent technological advances. This unit will conclude with some recent applications of two-photon microscopy that highlight the key advantages over confocal microscopy and the types of experiments which would benefit most from its application. PMID:23728746

ScanImage: flexible software for operating laser scanning microscopes.

PubMed

Pologruto, Thomas A; Sabatini, Bernardo L; Svoboda, Karel

2003-05-17

Laser scanning microscopy is a powerful tool for analyzing the structure and function of biological specimens. Although numerous commercial laser scanning microscopes exist, some of the more interesting and challenging applications demand custom design. A major impediment to custom design is the difficulty of building custom data acquisition hardware and writing the complex software required to run the laser scanning microscope. We describe a simple, software-based approach to operating a laser scanning microscope without the need for custom data acquisition hardware. Data acquisition and control of laser scanning are achieved through standard data acquisition boards. The entire burden of signal integration and image processing is placed on the CPU of the computer. We quantitate the effectiveness of our data acquisition and signal conditioning algorithm under a variety of conditions. We implement our approach in an open source software package (ScanImage) and describe its functionality. We present ScanImage, software to run a flexible laser scanning microscope that allows easy custom design.

Growth of carbon nanofibers on tipless cantilevers: process development and applications in scanning probe microscopy

NASA Astrophysics Data System (ADS)

Cui, Hongtao; Kalinin, Sergei; Yang, Xiaojing; Lowndes, Douglas

2005-03-01

Carbon nanofibers (CNFs) are grown on tipless cantilevers as probe tips for scanning probe microscopy. A catalyst dot pattern is formed on the surface of the tipless cantilever using electron beam lithography and CNF growth is performed in a direct-current plasma enhanced chemical vapor deposition reactor. Because the CNF is aligned with the electric field near the edge of the cantilever during growth, it is tilted with respect to the cantilever surface, which compensates partially for the probe tilt introduced when used in scanning probe microscopy. CNFs with different shapes and tip radii can be produced by variation of experimental conditions. The tip geometries of the CNF probes are defined by their catalyst particles, whose magnetic nature also imparts a capability for imaging magnetic samples. We have demonstrated their use in both atomic force and magnetic force surface imaging. These probe tips may provide information on magnetic phenomena at the nanometer scale in connection with the drive for ever-increasing storage density of magnetic hard disks.

Imaging interactions of metal oxide nanoparticles with macrophage cells by ultra-high resolution scanning electron microscopy techniques†

PubMed Central

Plascencia-Villa, Germán; Starr, Clarise R.; Armstrong, Linda S.; Ponce, Arturo

2016-01-01

Use of engineered metal oxide nanoparticles in a plethora of biological applications and custom products has warned about some possible dose-dependent cytotoxic effects. Macrophages are key components of the innate immune system used to study possible toxic effects and internalization of different nanoparticulate materials. In this work, ultra-high resolution field emission scanning electron microscopy (FE-SEM) was used to offer new insights into the dynamical processes of interaction of nanomaterials with macrophage cells dosed with different concentrations of metal oxide nanoparticles (CeO2, TiO2 and ZnO). The versatility of FE-SEM has allowed obtaining a detailed characterization of processes of adsorption and endocytosis of nanoparticles, by using advanced analytical and imaging techniques on complete unstained uncoated cells, including secondary electron imaging, high-sensitive backscattered electron imaging, X-ray microanalysis and stereoimaging. Low voltage BF/DF-STEM confirmed nanoparticle adsorption and internalization into endosomes of CeO2 and TiO2, whereas ZnO develop apoptosis after 24 h of interaction caused by dissolution and invasion of cell nucleus. Ultra-high resolution scanning electron microscopy techniques provided new insights into interactions of inorganic nanoparticles with macrophage cells with high spatial resolution. PMID:23023106

EFM data mapped into 2D images of tip-sample contact potential difference and capacitance second derivative.

PubMed

Lilliu, S; Maragliano, C; Hampton, M; Elliott, M; Stefancich, M; Chiesa, M; Dahlem, M S; Macdonald, J E

2013-11-27

We report a simple technique for mapping Electrostatic Force Microscopy (EFM) bias sweep data into 2D images. The method allows simultaneous probing, in the same scanning area, of the contact potential difference and the second derivative of the capacitance between tip and sample, along with the height information. The only required equipment consists of a microscope with lift-mode EFM capable of phase shift detection. We designate this approach as Scanning Probe Potential Electrostatic Force Microscopy (SPP-EFM). An open-source MATLAB Graphical User Interface (GUI) for images acquisition, processing and analysis has been developed. The technique is tested with Indium Tin Oxide (ITO) and with poly(3-hexylthiophene) (P3HT) nanowires for organic transistor applications.

How precise can atoms of a nanocluster be located in 3D using a tilt series of scanning transmission electron microscopy images?

PubMed

Alania, M; De Backer, A; Lobato, I; Krause, F F; Van Dyck, D; Rosenauer, A; Van Aert, S

2017-10-01

In this paper, we investigate how precise atoms of a small nanocluster can ultimately be located in three dimensions (3D) from a tilt series of images acquired using annular dark field (ADF) scanning transmission electron microscopy (STEM). Therefore, we derive an expression for the statistical precision with which the 3D atomic position coordinates can be estimated in a quantitative analysis. Evaluating this statistical precision as a function of the microscope settings also allows us to derive the optimal experimental design. In this manner, the optimal angular tilt range, required electron dose, optimal detector angles, and number of projection images can be determined. Copyright © 2016 Elsevier B.V. All rights reserved.

Lateral resolution improvement in scanning nonlinear dielectric microscopy by measuring super-higher-order nonlinear dielectric constants

NASA Astrophysics Data System (ADS)

Chinone, N.; Yamasue, K.; Hiranaga, Y.; Honda, K.; Cho, Y.

2012-11-01

Scanning nonlinear dielectric microscopy (SNDM) can be used to visualize polarization distributions in ferroelectric materials and dopant profiles in semiconductor devices. Without using a special sharp tip, we achieved an improved lateral resolution in SNDM through the measurement of super-higher-order nonlinearity up to the fourth order. We observed a multidomain single crystal congruent LiTaO3 (CLT) sample, and a cross section of a metal-oxide-semiconductor (MOS) field-effect-transistor (FET). The imaged domain boundaries of the CLT were narrower in the super-higher-order images than in the conventional image. Compared to the conventional method, the super-higher-order method resolved the more detailed structure of the MOSFET.

Imaging inflammation in mouse colon using a rapid stage-scanning confocal fluorescence microscope

NASA Astrophysics Data System (ADS)

Saldua, Meagan A.; Olsovsky, Cory A.; Callaway, Evelyn S.; Chapkin, Robert S.; Maitland, Kristen C.

2012-01-01

Large area confocal microscopy may provide fast, high-resolution image acquisition for evaluation of tissue in pre-clinical studies with reduced tissue processing in comparison to histology. We present a rapid beam and stage-scanning confocal fluorescence microscope to image cellular and tissue features along the length of the entire excised mouse colon. The beam is scanned at 8,333 lines/sec by a polygon scanning mirror while the specimen is scanned in the orthogonal axis by a motorized translation stage with a maximum speed of 7 mm/sec. A single 1×60 mm2 field of view image spanning the length of the mouse colon is acquired in 10 s. Z-projection images generated from axial image stacks allow high resolution imaging of the surface of non-flat specimens. In contrast to the uniform size, shape, and distribution of colon crypts in confocal images of normal colon, confocal images of chronic bowel inflammation exhibit heterogeneous tissue structure with localized severe crypt distortion.

Design and construction of a cost-efficient Arduino-based mirror galvanometer system for scanning optical microscopy

NASA Astrophysics Data System (ADS)

Hsu, Jen-Feng; Dhingra, Shonali; D'Urso, Brian

2017-01-01

Mirror galvanometer systems (galvos) are commonly employed in research and commercial applications in areas involving laser imaging, laser machining, laser-light shows, and others. Here, we present a robust, moderate-speed, and cost-efficient home-built galvo system. The mechanical part of this design consists of one mirror, which is tilted around two axes with multiple surface transducers. We demonstrate the ability of this galvo by scanning the mirror using a computer, via a custom driver circuit. The performance of the galvo, including scan range, noise, linearity, and scan speed, is characterized. As an application, we show that this galvo system can be used in a confocal scanning microscopy system.

Scanning electron microscopy imaging of dislocations in bulk materials, using electron channeling contrast.

PubMed

Crimp, Martin A

2006-05-01

The imaging and characterization of dislocations is commonly carried out by thin foil transmission electron microscopy (TEM) using diffraction contrast imaging. However, the thin foil approach is limited by difficult sample preparation, thin foil artifacts, relatively small viewable areas, and constraints on carrying out in situ studies. Electron channeling imaging of electron channeling contrast imaging (ECCI) offers an alternative approach for imaging crystalline defects, including dislocations. Because ECCI is carried out with field emission gun scanning electron microscope (FEG-SEM) using bulk specimens, many of the limitations of TEM thin foil analysis are overcome. This paper outlines the development of electron channeling patterns and channeling imaging to the current state of the art. The experimental parameters and set up necessary to carry out routine channeling imaging are reviewed. A number of examples that illustrate some of the advantages of ECCI over thin foil TEM are presented along with a discussion of some of the limitations on carrying out channeling contrast analysis of defect structures. Copyright (c) 2006 Wiley-Liss, Inc.

The role of gas in determining image quality and resolution during in situ scanning transmission electron microscopy experiments

DOE PAGES

Zhu, Yuanyuan; Browning, Nigel D.

2017-05-24

As gas-solid heterogeneous catalytic reactions are molecular in nature, a full mechanistic understanding of the process requires atomic scale characterization under realistic operating conditions. While atomic resolution imaging has become a routine in modern high-vacuum (scanning) transmission electron microscopy ((S)TEM), both image quality and resolution nominally degrade when reaction gases are introduced. In this work, we systematically assess the effects of different gases at various pressures on the quality and resolution of images obtained at room temperature in the annular dark field STEM imaging mode using a differentially pumped (DP) gas cell. This imaging mode is largely free from inelasticmore » scattering effects induced by the presence of gases and retains good imaging properties over a wide range of gas mass/pressures. Furthermore, we demonstrate the application of the ESTEM with atomic resolution images of a complex oxide alkane oxidation catalyst MoVNbTeOx (M1) immersed in light and heavy gas environments.« less

An instrument for 3D x-ray nano-imaging

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

Holler, M.; Raabe, J.; Diaz, A.

We present an instrument dedicated to 3D scanning x-ray microscopy, allowing a sample to be precisely scanned through a beam while the angle of x-ray incidence can be changed. The position of the sample is controlled with respect to the beam-defining optics by laser interferometry. The instrument achieves a position stability better than 10 nm standard deviation. The instrument performance is assessed using scanning x-ray diffraction microscopy and we demonstrate a resolution of 18 nm in 2D imaging of a lithographic test pattern while the beam was defined by a pinhole of 3 {mu}m in diameter. In 3D on amore » test object of copper interconnects of a microprocessor, a resolution of 53 nm is achieved.« less

Fast two-dimensional grid and transmission X-ray microscopy scanning methods for visualizing and characterizing protein crystals

PubMed Central

Wojdyla, Justyna Aleksandra; Panepucci, Ezequiel; Martiel, Isabelle; Ebner, Simon; Huang, Chia-Ying; Caffrey, Martin; Bunk, Oliver; Wang, Meitian

2016-01-01

A fast continuous grid scan protocol has been incorporated into the Swiss Light Source (SLS) data acquisition and analysis software suite on the macromolecular crystallography (MX) beamlines. Its combination with fast readout single-photon counting hybrid pixel array detectors (PILATUS and EIGER) allows for diffraction-based identification of crystal diffraction hotspots and the location and centering of membrane protein microcrystals in the lipid cubic phase (LCP) in in meso in situ serial crystallography plates and silicon nitride supports. Diffraction-based continuous grid scans with both still and oscillation images are supported. Examples that include a grid scan of a large (50 nl) LCP bolus and analysis of the resulting diffraction images are presented. Scanning transmission X-ray microscopy (STXM) complements and benefits from fast grid scanning. STXM has been demonstrated at the SLS beamline X06SA for near-zero-dose detection of protein crystals mounted on different types of sample supports at room and cryogenic temperatures. Flash-cooled crystals in nylon loops were successfully identified in differential and integrated phase images. Crystals of just 10 µm thickness were visible in integrated phase images using data collected with the EIGER detector. STXM offers a truly low-dose method for locating crystals on solid supports prior to diffraction data collection at both synchrotron microfocusing and free-electron laser X-ray facilities. PMID:27275141

Alternating current scanning electrochemical microscopy with simultaneous fast-scan cyclic voltammetry.

PubMed

Koch, Jason A; Baur, Melinda B; Woodall, Erica L; Baur, John E

2012-11-06

Fast-scan cyclic voltammetry (FSCV) is combined with alternating current scanning electrochemical microscopy (AC-SECM) for simultaneous measurements of impedance and faradaic current. Scan rates of 10-1000 V s(-1) were used for voltammetry, while a high-frequency (100 kHz), low-amplitude (10 mV rms) sine wave was added to the voltammetric waveform for the ac measurement. Both a lock-in amplifier and an analog circuit were used to measure the amplitude of the resultant ac signal. The effect of the added sine wave on the voltammetry at a carbon fiber electrode was investigated and found to have negligible effect. The combined FSCV and ac measurements were used to provide simultaneous chemical and topographical information about a substrate using a single carbon fiber probe. The technique is demonstrated in living cell culture, where cellular respiration and topography were simultaneously imaged without the addition of a redox mediator. This approach promises to be useful for the topographical and multidimensional chemical imaging of substrates.

Simulations For Investigating the Contrast Mechanism of Biological Cells with High Frequency Scanning Acoustic Microscopy

NASA Astrophysics Data System (ADS)

Juntarapaso, Yada

Scanning Acoustic Microscopy (SAM) is one of the most powerful techniques for nondestructive evaluation and it is a promising tool for characterizing the elastic properties of biological tissues/cells. Exploring a single cell is important since there is a connection between single cell biomechanics and human cancer. Scanning acoustic microscopy (SAM) has been accepted and extensively utilized for acoustical cellular and tissue imaging including measurements of the mechanical and elastic properties of biological specimens. SAM provides superb advantages in that it is non-invasive, can measure mechanical properties of biological cells or tissues, and fixation/chemical staining is not necessary. The first objective of this research is to develop a program for simulating the images and contrast mechanism obtained by high-frequency SAM. Computer simulation algorithms based on MatlabRTM were built for simulating the images and contrast mechanisms. The mechanical properties of HeLa and MCF-7 cells were computed from the measurement data of the output signal amplitude as a function of distance from the focal planes of the acoustics lens which is known as V(z) . Algorithms for simulating V(z) responses involved the calculation of the reflectance function and were created based on ray theory and wave theory. The second objective is to design transducer arrays for SAM. Theoretical simulations based on Field II(c) programs of the high frequency ultrasound array designs were performed to enhance image resolution and volumetric imaging capabilities. Phased array beam forming and dynamic apodization and focusing were employed in the simulations. The new transducer array design will be state-of-the-art in improving the performance of SAM by electronic scanning and potentially providing a 4-D image of the specimen.

Nonlinear laser scanning microscopy of human vocal folds.

PubMed

Miri, Amir K; Tripathy, Umakanta; Mongeau, Luc; Wiseman, Paul W

2012-02-01

The purpose of this work was to apply nonlinear laser scanning microscopy (NLSM) for visualizing the morphology of extracellular matrix proteins within human vocal folds. This technique may potentially assist clinicians in making rapid diagnoses of vocal fold tissue disease or damage. Microstructural characterization based on NLSM provides valuable information for better understanding molecular mechanisms and tissue structure. Experimental, ex vivo human vocal fold. A custom-built multimodal nonlinear laser scanning microscope was used to scan fibrillar proteins in three 4% formaldehyde-fixed cadaveric samples. Collagen and elastin, key extracellular matrix proteins in the vocal fold lamina propria, were imaged by two nonlinear microscopy modalities: second harmonic generation (SHG) and two-photon fluorescence (TPF), respectively. An experimental protocol was introduced to characterize the geometrical properties of the imaged fibrous proteins. NLSM revealed the biomorphology of the human vocal fold fibrous proteins. No photobleaching was observed for the incident laser power of ∼60 mW before the excitation objective. Types I and III fibrillar collagen were imaged without label in the tissue by intrinsic SHG. Imaging while rotating the incident laser light-polarization direction confirmed a helical shape for the collagen fibers. The amplitude, periodicity, and overall orientation were then computed for the helically distributed collagen network. The elastin network was simultaneously imaged via TPF and found to have a basket-like structure. In some regions, particularly close to the epithelium, colocalization of both extracellular matrix components were observed. A benchmark study is presented for quantitative real-time, ex vivo, NLSM imaging of the extracellular macromolecules in human vocal fold lamina propria. The results are promising for clinical applications. Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.

Applications and challenges of digital pathology and whole slide imaging.

PubMed

Higgins, C

2015-07-01

Virtual microscopy is a method for digitizing images of tissue on glass slides and using a computer to view, navigate, change magnification, focus and mark areas of interest. Virtual microscope systems (also called digital pathology or whole slide imaging systems) offer several advantages for biological scientists who use slides as part of their general, pharmaceutical, biotechnology or clinical research. The systems usually are based on one of two methodologies: area scanning or line scanning. Virtual microscope systems enable automatic sample detection, virtual-Z acquisition and creation of focal maps. Virtual slides are layered with multiple resolutions at each location, including the highest resolution needed to allow more detailed review of specific regions of interest. Scans may be acquired at 2, 10, 20, 40, 60 and 100 × or a combination of magnifications to highlight important detail. Digital microscopy starts when a slide collection is put into an automated or manual scanning system. The original slides are archived, then a server allows users to review multilayer digital images of the captured slides either by a closed network or by the internet. One challenge for adopting the technology is the lack of a universally accepted file format for virtual slides. Additional challenges include maintaining focus in an uneven sample, detecting specimens accurately, maximizing color fidelity with optimal brightness and contrast, optimizing resolution and keeping the images artifact-free. There are several manufacturers in the field and each has not only its own approach to these issues, but also its own image analysis software, which provides many options for users to enhance the speed, quality and accuracy of their process through virtual microscopy. Virtual microscope systems are widely used and are trusted to provide high quality solutions for teleconsultation, education, quality control, archiving, veterinary medicine, research and other fields.

Vibration compensation for high speed scanning tunneling microscopy

NASA Astrophysics Data System (ADS)

Croft, D.; Devasia, S.

1999-12-01

Low scanning speed is a fundamental limitation of scanning tunneling microscopes (STMs), making real time imaging of surface processes and nanofabrication impractical. The effective scanning bandwidth is currently limited by the smallest resonant vibrational frequency of the piezobased positioning system (i.e., scanner) used in the STM. Due to this limitation, the acquired images are distorted during high speed operations. In practice, the achievable scan rates are much less than 1/10th of the resonant vibrational frequency of the STM scanner. To alleviate the scanning speed limitation, this article describes an inversion-based approach that compensates for the structural vibrations in the scanner and thus, allows STM imaging at high scanning speeds (relative to the smallest resonant vibrational frequency). Experimental results are presented to show the increase in scanning speeds achievable by applying the vibration compensation methods.

Validation of Digital Microscopy Compared With Light Microscopy for the Diagnosis of Canine Cutaneous Tumors.

PubMed

Bertram, Christof A; Gurtner, Corinne; Dettwiler, Martina; Kershaw, Olivia; Dietert, Kristina; Pieper, Laura; Pischon, Hannah; Gruber, Achim D; Klopfleisch, Robert

2018-07-01

Integration of new technologies, such as digital microscopy, into a highly standardized laboratory routine requires the validation of its performance in terms of reliability, specificity, and sensitivity. However, a validation study of digital microscopy is currently lacking in veterinary pathology. The aim of the current study was to validate the usability of digital microscopy in terms of diagnostic accuracy, speed, and confidence for diagnosing and differentiating common canine cutaneous tumor types and to compare it to classical light microscopy. Therefore, 80 histologic sections including 17 different skin tumor types were examined twice as glass slides and twice as digital whole-slide images by 6 pathologists with different levels of experience at 4 time points. Comparison of both methods found digital microscopy to be noninferior for differentiating individual tumor types within the category epithelial and mesenchymal tumors, but diagnostic concordance was slightly lower for differentiating individual round cell tumor types by digital microscopy. In addition, digital microscopy was associated with significantly shorter diagnostic time, but diagnostic confidence was lower and technical quality was considered inferior for whole-slide images compared with glass slides. Of note, diagnostic performance for whole-slide images scanned at 200× magnification was noninferior in diagnostic performance for slides scanned at 400×. In conclusion, digital microscopy differs only minimally from light microscopy in few aspects of diagnostic performance and overall appears adequate for the diagnosis of individual canine cutaneous tumors with minor limitations for differentiating individual round cell tumor types and grading of mast cell tumors.

Near Field Imaging of Charge Transport in Gallium Nitride and Zinc Oxide Nanostructures

DTIC Science & Technology

2010-12-01

distribution of recombination luminescence . While researching the diffusion lengths of these structures, the author also observed that many of these... diffusion length of these structures can be extracted. E. NEAR FIELD IMAGING WITH NEAR FIELD SCANNING OPTICAL MICROSCOPY Near field scanning optical...composite AFM/NSOM images and the slope analysis to extract Ld, the minority carrier diffusion length , as described in Chapter 3. In all cases, excitation

Knowledge Extraction from Atomically Resolved Images.

PubMed

Vlcek, Lukas; Maksov, Artem; Pan, Minghu; Vasudevan, Rama K; Kalinin, Sergei V

2017-10-24

Tremendous strides in experimental capabilities of scanning transmission electron microscopy and scanning tunneling microscopy (STM) over the past 30 years made atomically resolved imaging routine. However, consistent integration and use of atomically resolved data with generative models is unavailable, so information on local thermodynamics and other microscopic driving forces encoded in the observed atomic configurations remains hidden. Here, we present a framework based on statistical distance minimization to consistently utilize the information available from atomic configurations obtained from an atomically resolved image and extract meaningful physical interaction parameters. We illustrate the applicability of the framework on an STM image of a FeSe x Te 1-x superconductor, with the segregation of the chalcogen atoms investigated using a nonideal interacting solid solution model. This universal method makes full use of the microscopic degrees of freedom sampled in an atomically resolved image and can be extended via Bayesian inference toward unbiased model selection with uncertainty quantification.

Analysis of STM images with pure and CO-functionalized tips: A first-principles and experimental study

NASA Astrophysics Data System (ADS)

Gustafsson, Alexander; Okabayashi, Norio; Peronio, Angelo; Giessibl, Franz J.; Paulsson, Magnus

2017-08-01

We describe a first-principles method to calculate scanning tunneling microscopy (STM) images, and compare the results to well-characterized experiments combining STM with atomic force microscopy (AFM). The theory is based on density functional theory with a localized basis set, where the wave functions in the vacuum gap are computed by propagating the localized-basis wave functions into the gap using a real-space grid. Constant-height STM images are computed using Bardeen's approximation method, including averaging over the reciprocal space. We consider copper adatoms and single CO molecules adsorbed on Cu(111), scanned with a single-atom copper tip with and without CO functionalization. The calculated images agree with state-of-the-art experiments, where the atomic structure of the tip apex is determined by AFM. The comparison further allows for detailed interpretation of the STM images.

Optical coherence microscopy as a novel, non-invasive method for the 4D live imaging of early mammalian embryos.

PubMed

Karnowski, Karol; Ajduk, Anna; Wieloch, Bartosz; Tamborski, Szymon; Krawiec, Krzysztof; Wojtkowski, Maciej; Szkulmowski, Maciej

2017-06-23

Imaging of living cells based on traditional fluorescence and confocal laser scanning microscopy has delivered an enormous amount of information critical for understanding biological processes in single cells. However, the requirement for a high numerical aperture and fluorescent markers still limits researchers' ability to visualize the cellular architecture without causing short- and long-term photodamage. Optical coherence microscopy (OCM) is a promising alternative that circumvents the technical limitations of fluorescence imaging techniques and provides unique access to fundamental aspects of early embryonic development, without the requirement for sample pre-processing or labeling. In the present paper, we utilized the internal motion of cytoplasm, as well as custom scanning and signal processing protocols, to effectively reduce the speckle noise typical for standard OCM and enable high-resolution intracellular time-lapse imaging. To test our imaging system we used mouse and pig oocytes and embryos and visualized them through fertilization and the first embryonic division, as well as at selected stages of oogenesis and preimplantation development. Because all morphological and morphokinetic properties recorded by OCM are believed to be biomarkers of oocyte/embryo quality, OCM may represent a new chapter in imaging-based preimplantation embryo diagnostics.

Functional connectivity in the mouse brain imaged by B-mode photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Nasiriavanaki, Mohammadreza; Xing, Wenxin; Xia, Jun; Wang, Lihong V.

2014-03-01

The increasing use of mouse models for human brain disease studies, coupled with the fact that existing functional imaging modalities cannot be easily applied to mice, presents an emerging need for a new functional imaging modality. Utilizing acoustic-resolution photoacoustic microscopy (AR-PAM), we imaged spontaneous cerebral hemodynamic fluctuations and their associated functional connections in the mouse brain. The images were acquired noninvasively in B-scan mode with a fast frame rate, a large field of view, and a high spatial resolution. At a location relative to the bregma 0, correlations were investigated inter-hemispherically between bilaterally homologous regions, as well as intra-hemispherically within the same functional regions. The functional connectivity in different functional regions was studied. The locations of these regions agreed well with the Paxinos mouse brain atlas. The functional connectivity map obtained in this study can then be used in the investigation of brain disorders such as stroke, Alzheimer's, schizophrenia, multiple sclerosis, autism, and epilepsy. Our experiments show that photoacoustic microscopy is capable to detect connectivities between different functional regions in B-scan mode, promising a powerful functional imaging modality for future brain research.

Spatially-controlled illumination with rescan confocal microscopy enhances image quality, resolution and reduces photodamage

NASA Astrophysics Data System (ADS)

Krishnaswami, Venkataraman; De Luca, Giulia M. R.; Breedijk, Ronald M. P.; Van Noorden, Cornelis J. F.; Manders, Erik M. M.; Hoebe, Ron A.

2017-02-01

Fluorescence microscopy is an important tool in biomedical imaging. An inherent trade-off lies between image quality and photodamage. Recently, we have introduced rescan confocal microscopy (RCM) that improves the lateral resolution of a confocal microscope down to 170 nm. Previously, we have demonstrated that with controlled-light exposure microscopy, spatial control of illumination reduces photodamage without compromising image quality. Here, we show that the combination of these two techniques leads to high resolution imaging with reduced photodamage without compromising image quality. Implementation of spatially-controlled illumination was carried out in RCM using a line scanning-based approach. Illumination is spatially-controlled for every line during imaging with the help of a prediction algorithm that estimates the spatial profile of the fluorescent specimen. The estimation is based on the information available from previously acquired line images. As a proof-of-principle, we show images of N1E-115 neuroblastoma cells, obtained by this new setup with reduced illumination dose, improved resolution and without compromising image quality.

Identification of nanoparticles and nanosystems in biological matrices with scanning probe microscopy.

PubMed

Angeloni, Livia; Reggente, Melania; Passeri, Daniele; Natali, Marco; Rossi, Marco

2018-04-17

Identification of nanoparticles and nanosystems into cells and biological matrices is a hot research topic in nanobiotechnologies. Because of their capability to map physical properties (mechanical, electric, magnetic, chemical, or optical), several scanning probe microscopy based techniques have been proposed for the subsurface detection of nanomaterials in biological systems. In particular, atomic force microscopy (AFM) can be used to reveal stiff nanoparticles in cells and other soft biomaterials by probing the sample mechanical properties through the acquisition of local indentation curves or through the combination of ultrasound-based methods, like contact resonance AFM (CR-AFM) or scanning near field ultrasound holography. Magnetic force microscopy can detect magnetic nanoparticles and other magnetic (bio)materials in nonmagnetic biological samples, while electric force microscopy, conductive AFM, and Kelvin probe force microscopy can reveal buried nanomaterials on the basis of the differences between their electric properties and those of the surrounding matrices. Finally, scanning near field optical microscopy and tip-enhanced Raman spectroscopy can visualize buried nanostructures on the basis of their optical and chemical properties. Despite at a still early stage, these methods are promising for detection of nanomaterials in biological systems as they could be truly noninvasive, would not require destructive and time-consuming specific sample preparation, could be performed in vitro, on alive samples and in water or physiological environment, and by continuously imaging the same sample could be used to dynamically monitor the diffusion paths and interaction mechanisms of nanomaterials into cells and biological systems. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology. © 2018 Wiley Periodicals, Inc.

Precision controlled atomic resolution scanning transmission electron microscopy using spiral scan pathways

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

Sang, Xiahan; Lupini, Andrew R.; Ding, Jilai

Atomic-resolution imaging in an aberration-corrected scanning transmission electron microscope (STEM) can enable direct correlation between atomic structure and materials functionality. The fast and precise control of the STEM probe is, however, challenging because the true beam location deviates from the assigned location depending on the properties of the deflectors. To reduce these deviations, i.e. image distortions, we use spiral scanning paths, allowing precise control of a sub-Å sized electron probe within an aberration-corrected STEM. Although spiral scanning avoids the sudden changes in the beam location (fly-back distortion) present in conventional raster scans, it is not distortion-free. “Archimedean” spirals, with amore » constant angular frequency within each scan, are used to determine the characteristic response at different frequencies. We then show that such characteristic functions can be used to correct image distortions present in more complicated constant linear velocity spirals, where the frequency varies within each scan. Through the combined application of constant linear velocity scanning and beam path corrections, spiral scan images are shown to exhibit less scan distortion than conventional raster scan images. The methodology presented here will be useful for in situ STEM imaging at higher temporal resolution and for imaging beam sensitive materials.« less

Precision controlled atomic resolution scanning transmission electron microscopy using spiral scan pathways

DOE PAGES

Sang, Xiahan; Lupini, Andrew R.; Ding, Jilai; ...

2017-03-08

Atomic-resolution imaging in an aberration-corrected scanning transmission electron microscope (STEM) can enable direct correlation between atomic structure and materials functionality. The fast and precise control of the STEM probe is, however, challenging because the true beam location deviates from the assigned location depending on the properties of the deflectors. To reduce these deviations, i.e. image distortions, we use spiral scanning paths, allowing precise control of a sub-Å sized electron probe within an aberration-corrected STEM. Although spiral scanning avoids the sudden changes in the beam location (fly-back distortion) present in conventional raster scans, it is not distortion-free. “Archimedean” spirals, with amore » constant angular frequency within each scan, are used to determine the characteristic response at different frequencies. We then show that such characteristic functions can be used to correct image distortions present in more complicated constant linear velocity spirals, where the frequency varies within each scan. Through the combined application of constant linear velocity scanning and beam path corrections, spiral scan images are shown to exhibit less scan distortion than conventional raster scan images. The methodology presented here will be useful for in situ STEM imaging at higher temporal resolution and for imaging beam sensitive materials.« less

Evaluating bandgap distributions of carbon nanotubes via scanning electron microscopy imaging of the Schottky barriers.

PubMed

He, Yujun; Zhang, Jin; Li, Dongqi; Wang, Jiangtao; Wu, Qiong; Wei, Yang; Zhang, Lina; Wang, Jiaping; Liu, Peng; Li, Qunqing; Fan, Shoushan; Jiang, Kaili

2013-01-01

We show that the Schottky barrier at the metal-single walled carbon nanotube (SWCNT) contact can be clearly observed in scanning electron microscopy (SEM) images as a bright contrast segment with length up to micrometers due to the space charge distribution in the depletion region. The lengths of the charge depletion increase with the diameters of semiconducting SWCNTs (s-SWCNTs) when connected to one metal electrode, which enables direct and efficient evaluation of the bandgap distributions of s-SWCNTs. Moreover, this approach can also be applied for a wide variety of semiconducting nanomaterials, adding a new function to conventional SEM.

Gold nanoparticle uptake in whole cells in liquid examined by environmental scanning electron microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2014-02-01

The size of gold nanoparticles (AuNPs) can influence various aspects of their cellular uptake. Light microscopy is not capable of resolving most AuNPs, while electron microscopy (EM) is not practically capable of acquiring the necessary statistical data from many cells and the results may suffer from various artifacts. Here, we demonstrate the use of a fast EM method for obtaining high-resolution data from a much larger population of cells than is usually feasible with conventional EM. A549 (human lung carcinoma) cells were subjected to uptake protocols with 10, 15, or 30 nm diameter AuNPs with adsorbed serum proteins. After 20 min, 24 h, or 45 h, the cells were fixed and imaged in whole in a thin layer of liquid water with environmental scanning electron microscopy equipped with a scanning transmission electron microscopy detector. The fast preparation and imaging of 145 whole cells in liquid allowed collection of nanoscale data within an exceptionally small amount of time of ~80 h. Analysis of 1,041 AuNP-filled vesicles showed that the long-term AuNP storing lysosomes increased their average size by 80 nm when AuNPs with 30 nm diameter were uptaken, compared to lysosomes of cells incubated with AuNPs of 10 and 15 nm diameter.

Big, Deep, and Smart Data in Scanning Probe Microscopy

DOE PAGES

Kalinin, Sergei V.; Strelcov, Evgheni; Belianinov, Alex; ...

2016-09-27

Scanning probe microscopy techniques open the door to nanoscience and nanotechnology by enabling imaging and manipulation of structure and functionality of matter on nanometer and atomic scales. We analyze the discovery process by SPM in terms of information flow from tip-surface junction to the knowledge adoption by scientific community. Furthermore, we discuss the challenges and opportunities offered by merging of SPM and advanced data mining, visual analytics, and knowledge discovery technologies.

A history of scanning electron microscopy developments: towards "wet-STEM" imaging.

PubMed

Bogner, A; Jouneau, P-H; Thollet, G; Basset, D; Gauthier, C

2007-01-01

A recently developed imaging mode called "wet-STEM" and new developments in environmental scanning electron microscopy (ESEM) allows the observation of nano-objects suspended in a liquid phase, with a few manometers resolution and a good signal to noise ratio. The idea behind this technique is simply to perform STEM-in-SEM, that is SEM in transmission mode, in an environmental SEM. The purpose of the present contribution is to highlight the main advances that contributed to development of the wet-STEM technique. Although simple in principle, the wet-STEM imaging mode would have been limited before high brightness electron sources became available, and needed some progresses and improvements in ESEM. This new technique extends the scope of SEM as a high-resolution microscope, relatively cheap and widely available imaging tool, for a wider variety of samples.

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.

Random-access optical-resolution photoacoustic microscopy using a digital micromirror device

PubMed Central

Liang, Jinyang; Zhou, Yong; Winkler, Amy W.; Wang, Lidai; Maslov, Konstantin I.; Li, Chiye; Wang, Lihong V.

2013-01-01

We developed random-access optical-resolution photoacoustic microscopy using a digital micromirror device. This system can rapidly scan arbitrarily shaped regions of interest within a 40×40 μm2 imaging area with a lateral resolution of 3.6 μm. To identify a region of interest, a global structural image is first acquired, then the selected region is scanned. The random-access ability was demonstrated by imaging two static samples, a carbon fiber cross and a monolayer of red blood cells, with an acquisition rate up to 4 kilohertz. The system was then used to monitor blood flow in vivo in real time within user-selected capillaries in a mouse ear. By imaging only the capillary of interest, the frame rate was increased by up to 9.2 times. PMID:23903111

Random-access optical-resolution photoacoustic microscopy using a digital micromirror device.

PubMed

Liang, Jinyang; Zhou, Yong; Winkler, Amy W; Wang, Lidai; Maslov, Konstantin I; Li, Chiye; Wang, Lihong V

2013-08-01

We developed random-access optical-resolution photoacoustic microscopy using a digital micromirror device. This system can rapidly scan arbitrarily shaped regions of interest within a 40 μm×40 μm imaging area with a lateral resolution of 3.6 μm. To identify a region of interest, a global structural image is first acquired, then the selected region is scanned. The random-access ability was demonstrated by imaging two static samples, a carbon fiber cross and a monolayer of red blood cells, with an acquisition rate up to 4 kHz. The system was then used to monitor blood flow in vivo in real time within user-selected capillaries in a mouse ear. By imaging only the capillary of interest, the frame rate was increased by up to 9.2 times.

Adaptive optics for confocal laser scanning microscopy with adjustable pinhole

NASA Astrophysics Data System (ADS)

Yoo, Han Woong; van Royen, Martin E.; van Cappellen, Wiggert A.; Houtsmuller, Adriaan B.; Verhaegen, Michel; Schitter, Georg

2016-04-01

The pinhole plays an important role in confocal laser scanning microscopy (CLSM) for adaptive optics (AO) as well as in imaging, where the size of the pinhole denotes a trade-off between out-of-focus rejection and wavefront distortion. This contribution proposes an AO system for a commercial CLSM with an adjustable square pinhole to cope with such a trade-off. The proposed adjustable pinhole enables to calibrate the AO system and to evaluate the imaging performance. Experimental results with fluorescence beads on the coverslip and at a depth of 40 μm in the human hepatocellular carcinoma cell spheroid demonstrate that the proposed AO system can improve the image quality by the proposed calibration method. The proposed pinhole intensity ratio also indicates the image improvement by the AO correction in intensity as well as resolution.

Massively Parallel Rogue Cell Detection Using Serial Time-Encoded Amplified Microscopy of Inertially Ordered Cells in High-Throughput Flow

DTIC Science & Technology

2012-08-01

techniques and STEAM imager. It couples the high-speed capability of the STEAM imager and differential phase contrast imaging of DIC / Nomarski microscopy...On 10 TPE chips, we obtained 9 homogenous and strong bonds, the failed bond being due to operator error and presence of air bubbles in the TPE...instruments, structural dynamics, and microelectromechanical systems (MEMS) via laser-scanning surface vibrometry , and observation of biomechanical motility

A compilation of cold cases using scanning electron microscopy at the University of Rhode Island

NASA Astrophysics Data System (ADS)

Platek, Michael J.; Gregory, Otto J.

2015-10-01

Scanning electron microscopy combined with microchemical analysis has evolved into one of the most widely used instruments in forensic science today. In particular, the environmental scanning electron microscope (SEM) in conjunction with energy dispersive spectroscopy (EDS), has created unique opportunities in forensic science in regard to the examination of trace evidence; i.e. the examination of evidence without altering the evidence with conductive coatings, thereby enabling criminalists to solve cases that were previously considered unsolvable. Two cold cases were solved at URI using a JEOL 5900 LV SEM in conjunction with EDS. A cold case murder and a cold missing person case will be presented from the viewpoint of the microscopist and will include sample preparation, as well as image and chemical analysis of the trace evidence using electron microscopy and optical microscopy.

Electron tomography of HEK293T cells using scanning electron microscope-based scanning transmission electron microscopy.

PubMed

You, Yun-Wen; Chang, Hsun-Yun; Liao, Hua-Yang; Kao, Wei-Lun; Yen, Guo-Ji; Chang, Chi-Jen; Tsai, Meng-Hung; Shyue, Jing-Jong

2012-10-01

Based on a scanning electron microscope operated at 30 kV with a homemade specimen holder and a multiangle solid-state detector behind the sample, low-kV scanning transmission electron microscopy (STEM) is presented with subsequent electron tomography for three-dimensional (3D) volume structure. Because of the low acceleration voltage, the stronger electron-atom scattering leads to a stronger contrast in the resulting image than standard TEM, especially for light elements. Furthermore, the low-kV STEM yields less radiation damage to the specimen, hence the structure can be preserved. In this work, two-dimensional STEM images of a 1-μm-thick cell section with projection angles between ±50° were collected, and the 3D volume structure was reconstructed using the simultaneous iterative reconstructive technique algorithm with the TomoJ plugin for ImageJ, which are both public domain software. Furthermore, the cross-sectional structure was obtained with the Volume Viewer plugin in ImageJ. Although the tilting angle is constrained and limits the resulting structural resolution, slicing the reconstructed volume generated the depth profile of the thick specimen with sufficient resolution to examine cellular uptake of Au nanoparticles, and the final position of these nanoparticles inside the cell was imaged.

New Algorithm to Enable Construction and Display of 3D Structures from Scanning Probe Microscopy Images Acquired Layer-by-Layer.

PubMed

Deng, William Nanqiao; Wang, Shuo; Ventrici de Souza, Joao; Kuhl, Tonya L; Liu, Gang-Yu

2018-06-25

Scanning probe microscopy (SPM), such as atomic force microscopy (AFM), is widely known for high-resolution imaging of surface structures and nanolithography in two dimensions (2D), providing important physical insights into surface science and material science. This work reports a new algorithm to enable construction and display of layer-by-layer 3D structures from SPM images. The algorithm enables alignment of SPM images acquired during layer-by-layer deposition and removal of redundant features and faithfully constructs the deposited 3D structures. The display uses a "see-through" strategy to enable the structure of each layer to be visible. The results demonstrate high spatial accuracy as well as algorithm versatility; users can set parameters for reconstruction and display as per image quality and research needs. To the best of our knowledge, this method represents the first report to enable SPM technology for 3D imaging construction and display. The detailed algorithm is provided to facilitate usage of the same approach in any SPM software. These new capabilities support wide applications of SPM that require 3D image reconstruction and display, such as 3D nanoprinting and 3D additive and subtractive manufacturing and imaging.

Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology

PubMed Central

Sandell, Lisa L.; Kurosaka, Hiroshi; Trainor, Paul A.

2012-01-01

Here we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional widefield fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images of similar specimens produced by Scanning Electron Microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. PMID:22930523

Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology.

PubMed

Sandell, Lisa L; Kurosaka, Hiroshi; Trainor, Paul A

2012-11-01

Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. Copyright © 2012 Wiley Periodicals, Inc.

Optical resolution photoacoustic microscopy using novel high-repetition-rate passively Q-switched microchip and fiber lasers.

PubMed

Shi, Wei; Kerr, Shaun; Utkin, Ilya; Ranasinghesagara, Janaka; Pan, Lei; Godwal, Yogesh; Zemp, Roger J; Fedosejevs, Robert

2010-01-01

Optical-resolution photoacoustic microscopy (OR-PAM) is a novel imaging technology for visualizing optically absorbing superficial structures in vivo with lateral spatial resolution determined by optical focusing rather than acoustic detection. Since scanning of the illumination spot is required, OR-PAM imaging speed is limited by both scanning speed and laser pulse repetition rate. Unfortunately, lasers with high repetition rates and suitable pulse durations and energies are not widely available and can be cost-prohibitive and bulky. We are developing compact, passively Q-switched fiber and microchip laser sources for this application. The properties of these lasers are discussed, and pulse repetition rates up to 100 kHz are demonstrated. OR-PAM imaging was conducted using a previously developed photoacoustic probe, which enabled flexible scanning of the focused output of the lasers. Phantom studies demonstrate the ability to image with lateral spatial resolution of 7±2 μm with the microchip laser system and 15±5 μm with the fiber laser system. We believe that the high pulse repetition rates and the potentially compact and fiber-coupled nature of these lasers will prove important for clinical imaging applications where real-time imaging performance is essential.

STM, SECPM, AFM and Electrochemistry on Single Crystalline Surfaces

PubMed Central

Wolfschmidt, Holger; Baier, Claudia; Gsell, Stefan; Fischer, Martin; Schreck, Matthias; Stimming, Ulrich

2010-01-01

Scanning probe microscopy (SPM) techniques have had a great impact on research fields of surface science and nanotechnology during the last decades. They are used to investigate surfaces with scanning ranges between several 100 μm down to atomic resolution. Depending on experimental conditions, and the interaction forces between probe and sample, different SPM techniques allow mapping of different surface properties. In this work, scanning tunneling microscopy (STM) in air and under electrochemical conditions (EC-STM), atomic force microscopy (AFM) in air and scanning electrochemical potential microscopy (SECPM) under electrochemical conditions, were used to study different single crystalline surfaces in electrochemistry. Especially SECPM offers potentially new insights into the solid-liquid interface by providing the possibility to image the potential distribution of the surface, with a resolution that is comparable to STM. In electrocatalysis, nanostructured catalysts supported on different electrode materials often show behavior different from their bulk electrodes. This was experimentally and theoretically shown for several combinations and recently on Pt on Au(111) towards fuel cell relevant reactions. For these investigations single crystals often provide accurate and well defined reference and support systems. We will show heteroepitaxially grown Ru, Ir and Rh single crystalline surface films and bulk Au single crystals with different orientations under electrochemical conditions. Image studies from all three different SPM methods will be presented and compared to electrochemical data obtained by cyclic voltammetry in acidic media. The quality of the single crystalline supports will be verified by the SPM images and the cyclic voltammograms. Furthermore, an outlook will be presented on how such supports can be used in electrocatalytic studies. PMID:28883327

Material State Awareness for Composites Part II: Precursor Damage Analysis and Quantification of Degraded Material Properties Using Quantitative Ultrasonic Image Correlation (QUIC)

PubMed Central

Patra, Subir; Banerjee, Sourav

2017-01-01

Material state awareness of composites using conventional Nondestructive Evaluation (NDE) method is limited by finding the size and the locations of the cracks and the delamination in a composite structure. To aid the progressive failure models using the slow growth criteria, the awareness of the precursor damage state and quantification of the degraded material properties is necessary, which is challenging using the current NDE methods. To quantify the material state, a new offline NDE method is reported herein. The new method named Quantitative Ultrasonic Image Correlation (QUIC) is devised, where the concept of microcontinuum mechanics is hybrid with the experimentally measured Ultrasonic wave parameters. This unique combination resulted in a parameter called Nonlocal Damage Entropy for the precursor awareness. High frequency (more than 25 MHz) scanning acoustic microscopy is employed for the proposed QUIC. Eight woven carbon-fiber-reinforced-plastic composite specimens were tested under fatigue up to 70% of their remaining useful life. During the first 30% of the life, the proposed nonlocal damage entropy is plotted to demonstrate the degradation of the material properties via awareness of the precursor damage state. Visual proofs for the precursor damage states are provided with the digital images obtained from the micro-optical microscopy, the scanning acoustic microscopy and the scanning electron microscopy. PMID:29258256

A CANDLE for a deeper in vivo insight

PubMed Central

Coupé, Pierrick; Munz, Martin; Manjón, Jose V; Ruthazer, Edward S; Louis Collins, D.

2012-01-01

A new Collaborative Approach for eNhanced Denoising under Low-light Excitation (CANDLE) is introduced for the processing of 3D laser scanning multiphoton microscopy images. CANDLE is designed to be robust for low signal-to-noise ratio (SNR) conditions typically encountered when imaging deep in scattering biological specimens. Based on an optimized non-local means filter involving the comparison of filtered patches, CANDLE locally adapts the amount of smoothing in order to deal with the noise inhomogeneity inherent to laser scanning fluorescence microscopy images. An extensive validation on synthetic data, images acquired on microspheres and in vivo images is presented. These experiments show that the CANDLE filter obtained competitive results compared to a state-of-the-art method and a locally adaptive optimized nonlocal means filter, especially under low SNR conditions (PSNR<8dB). Finally, the deeper imaging capabilities enabled by the proposed filter are demonstrated on deep tissue in vivo images of neurons and fine axonal processes in the Xenopus tadpole brain. PMID:22341767

An evaluation of a combined scanning probe and optical microscope for lunar regolith studies

NASA Astrophysics Data System (ADS)

Yang, S.; Pike, W. T.; Staufer, U.; Claus, D.; Rodenburg, J. M.

2011-12-01

The microscopic properties of the lunar regolith such as the shape, the surface texture and the size distribution are required for an understanding of both past surface processes and potential hazards for future human exploration [1]. To reveal the particle morphology at the sub micrometer scale, scanning-probe microscopy (SPM), first used on the 2008 Phoenix mission [1], is a proven approach; however, there are two main challenges for the measurement of lunar particles. Firstly, the SPM tip is liable to move particles during scanning, even when using the lower contact forces of the dynamic-mode imaging. Hence the particles need to be stabilised during imaging. Secondly, typically the AFM tip extends about 10 μm from its cantilever, so larger particles protruding more than this height above their substrates cannot be scanned completely. To immobilize particles and eliminate large particles during SPM scanning, micromachined Si substrates, which have been successfully applied in the Phoenix project for Mars investigation in 2008 [2], have been investigated for lunar analogue material. On these substrates micrometer pits are patterned and serve as traps to enhance the stability of the AFM scanning by grasping the particles. In addition, the diameter of pits can determine the size of dusts to be captured and reduce the adhesion for the larger dust and so eliminate the oversized particles. To extend the imaging range and assist in selecting scan areas for the SPM, we use a type of lensless optical imaging (LOM) which uses ptychographic diffractive imaging [3] to eliminate the restrictions and performance limitations of conventional focusing devices. As a reference, scanning electron microscopy (SEM) which minimizes particle-probe interactions and has the advantage of an extended depth of field, is employed to image the same particle fields at resolutions covering both the SPM and LOM. By comparing the differences and the similarities between SEM and LOM images, the ability of LOM for illuminating the details about the lunar particles sample, is demonstrated. The analysis of SEM and SPM images of the same particles of JSC-LunarA analogue soil reveals the potential of the SPM to obtain reliable microscopic images of lunar dusts including detailed morphology with the help of the micromachined Si substrates. [1] J. D. Carpenter, O. Angerer, M. Durante, D. Linnarson, W. T. Pike, "Life Sciences Investigations for ESA's First Lunar Lander," Earth, Moon, and Planets, Vol.107, pp. 11-23, 2010. [2] S. Vijendran, H.Sykulska, and W. T. Pike, "AFM investigation of Martian soil simulant on micromachined Si substrates," Journal of Microscopy, Vol.227, pp.236-245, Sep. 2007. [3] J.M. Rodenburg, "Ptychography and related diffractive imaging techniques," Advances in Imaging and Electron Physics, Vol.150, pp. 87-184, 2008

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

NASA Astrophysics Data System (ADS)

Ding, Chenliang; Wei, Jingsong

2016-01-01

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

Serial sectioning methods for 3D investigations in materials science.

PubMed

Zankel, Armin; Wagner, Julian; Poelt, Peter

2014-07-01

A variety of methods for the investigation and 3D representation of the inner structure of materials has been developed. In this paper, techniques based on slice and view using scanning microscopy for imaging are presented and compared. Three different methods of serial sectioning combined with either scanning electron or scanning ion microscopy or atomic force microscopy (AFM) were placed under scrutiny: serial block-face scanning electron microscopy, which facilitates an ultramicrotome built into the chamber of a variable pressure scanning electron microscope; three-dimensional (3D) AFM, which combines an (cryo-) ultramicrotome with an atomic force microscope, and 3D FIB, which delivers results by slicing with a focused ion beam. These three methods complement one another in many respects, e.g., in the type of materials that can be investigated, the resolution that can be obtained and the information that can be extracted from 3D reconstructions. A detailed review is given about preparation, the slice and view process itself, and the limitations of the methods and possible artifacts. Applications for each technique are also provided. Copyright © 2014 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.

Modular Scanning Confocal Microscope with Digital Image Processing.

PubMed

Ye, Xianjun; McCluskey, Matthew D

2016-01-01

In conventional confocal microscopy, a physical pinhole is placed at the image plane prior to the detector to limit the observation volume. In this work, we present a modular design of a scanning confocal microscope which uses a CCD camera to replace the physical pinhole for materials science applications. Experimental scans were performed on a microscope resolution target, a semiconductor chip carrier, and a piece of etched silicon wafer. The data collected by the CCD were processed to yield images of the specimen. By selecting effective pixels in the recorded CCD images, a virtual pinhole is created. By analyzing the image moments of the imaging data, a lateral resolution enhancement is achieved by using a 20 × / NA = 0.4 microscope objective at 532 nm laser wavelength.

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection

PubMed Central

Zhi, Yanan; Wang, Benquan; Yao, Xincheng

2016-01-01

Light microscopy plays a key role in biological studies and medical diagnosis. The spatial resolution of conventional optical microscopes is limited to approximately half the wavelength of the illumination light as a result of the diffraction limit. Several approaches—including confocal microscopy, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy, and structured illumination microscopy—have been established to achieve super-resolution imaging. However, none of these methods is suitable for the super-resolution ophthalmoscopy of retinal structures because of laser safety issues and inevitable eye movements. We recently experimentally validated virtually structured detection (VSD) as an alternative strategy to extend the diffraction limit. Without the complexity of structured illumination, VSD provides an easy, low-cost, and phase artifact–free strategy to achieve super-resolution in scanning laser microscopy. In this article we summarize the basic principles of the VSD method, review our demonstrated single-point and line-scan super-resolution systems, and discuss both technical challenges and the potential of VSD-based instrumentation for super-resolution ophthalmoscopy of the retina. PMID:27480461

Parallel detection experiment of fluorescence confocal microscopy using DMD.

PubMed

Wang, Qingqing; Zheng, Jihong; Wang, Kangni; Gui, Kun; Guo, Hanming; Zhuang, Songlin

2016-05-01

Parallel detection of fluorescence confocal microscopy (PDFCM) system based on Digital Micromirror Device (DMD) is reported in this paper in order to realize simultaneous multi-channel imaging and improve detection speed. DMD is added into PDFCM system, working to take replace of the single traditional pinhole in the confocal system, which divides the laser source into multiple excitation beams. The PDFCM imaging system based on DMD is experimentally set up. The multi-channel image of fluorescence signal of potato cells sample is detected by parallel lateral scanning in order to verify the feasibility of introducing the DMD into fluorescence confocal microscope. In addition, for the purpose of characterizing the microscope, the depth response curve is also acquired. The experimental result shows that in contrast to conventional microscopy, the DMD-based PDFCM system has higher axial resolution and faster detection speed, which may bring some potential benefits in the biology and medicine analysis. SCANNING 38:234-239, 2016. © 2015 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

High-Resolution Imaging of Polyethylene Glycol Coated Dendrimers via Combined Atomic Force and Scanning Tunneling Microscopy

PubMed Central

Zhong, Qian; Yin, Nai-Ning; Karsai, Arpad; da Rocha, Sandro R. P.; Liu, Gang-yu

2015-01-01

Dendrimers have shown great promise as drug delivery vehicles in recent years because they can be synthesized with designed size and functionalities for optimal transportation, targeting, and biocompatibility. One of the most well-known termini used for biocompatibility is polyethylene glycol (PEG), whose performance is affected by its actual conformation. However, the conformation of individual PEG bound to soft materials such as dendrimers has not been directly observed. Using atomic force microscopy (AFM) and scanning tunneling microscopy (STM), this work characterizes the structure adopted by PEGylated dendrimers with the highest resolution reported to date. AFM imaging enables visualization of the individual dendrimers, as well as the differentiation and characterization of the dendrimer core and PEG shell. STM provides direct imaging of the PEG extensions with high-resolution. Collectively, this investigation provides important insight into the structure of coated dendrimers, which is crucial for the design and development of better drug delivery vehicles. PMID:25685559

Revealing the correlation between real-space structure and chiral magnetic order at the atomic scale

NASA Astrophysics Data System (ADS)

Hauptmann, Nadine; Dupé, Melanie; Hung, Tzu-Chao; Lemmens, Alexander K.; Wegner, Daniel; Dupé, Bertrand; Khajetoorians, Alexander A.

2018-03-01

We image simultaneously the geometric, the electronic, and the magnetic structures of a buckled iron bilayer film that exhibits chiral magnetic order. We achieve this by combining spin-polarized scanning tunneling microscopy and magnetic exchange force microscopy (SPEX) to independently characterize the geometric as well as the electronic and magnetic structures of nonflat surfaces. This new SPEX imaging technique reveals the geometric height corrugation of the reconstruction lines resulting from strong strain relaxation in the bilayer, enabling the decomposition of the real-space from the electronic structure at the atomic level and the correlation with the resultant spin-spiral ground state. By additionally utilizing adatom manipulation, we reveal the chiral magnetic ground state of portions of the unit cell that were not previously imaged with spin-polarized scanning tunneling microscopy alone. Using density functional theory, we investigate the structural and electronic properties of the reconstructed bilayer and identify the favorable stoichiometry regime in agreement with our experimental result.

Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing.

PubMed

Moo, Eng Kuan; Abusara, Ziad; Abu Osman, Noor Azuan; Pingguan-Murphy, Belinda; Herzog, Walter

2013-08-09

Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in cell responses to mechanical stimuli that depend on the microscopic approach and the thresholding methods used and may result in contradictory interpretations. Copyright © 2013 Elsevier Ltd. All rights reserved.

Calibration-free quantitative surface topography reconstruction in scanning electron microscopy.

PubMed

Faber, E T; Martinez-Martinez, D; Mansilla, C; Ocelík, V; Hosson, J Th M De

2015-01-01

This work presents a new approach to obtain reliable surface topography reconstructions from 2D Scanning Electron Microscopy (SEM) images. In this method a set of images taken at different tilt angles are compared by means of digital image correlation (DIC). It is argued that the strength of the method lies in the fact that precise knowledge about the nature of the rotation (vector and/or magnitude) is not needed. Therefore, the great advantage is that complex calibrations of the measuring equipment are avoided. The paper presents the necessary equations involved in the methods, including derivations and solutions. The method is illustrated with examples of 3D reconstructions followed by a discussion on the relevant experimental parameters. Copyright © 2014 Elsevier B.V. All rights reserved.

Examination of enterotoxigenic Escherichia coli H10407 (colonization factor antigen I+) by scanning electron microscopy with conductive staining.

PubMed Central

Sherburne, R; Armstrong, G D

1989-01-01

We have used the scanning electron microscope to examine enterotoxigenic Escherichia coli H10407, which expresses colonization factor antigen I pili. The use of low accelerating voltages and conductive staining procedures allowed us to obtain images of colonization factor antigen I pili and other structural details which were obscured by conventional gold-coating techniques. Images PMID:2570062

Richardson-Lucy deconvolution as a general tool for combining images with complementary strengths.

PubMed

Ingaramo, Maria; York, Andrew G; Hoogendoorn, Eelco; Postma, Marten; Shroff, Hari; Patterson, George H

2014-03-17

We use Richardson-Lucy (RL) deconvolution to combine multiple images of a simulated object into a single image in the context of modern fluorescence microscopy techniques. RL deconvolution can merge images with very different point-spread functions, such as in multiview light-sheet microscopes,1, 2 while preserving the best resolution information present in each image. We show that RL deconvolution is also easily applied to merge high-resolution, high-noise images with low-resolution, low-noise images, relevant when complementing conventional microscopy with localization microscopy. We also use RL deconvolution to merge images produced by different simulated illumination patterns, relevant to structured illumination microscopy (SIM)3, 4 and image scanning microscopy (ISM). The quality of our ISM reconstructions is at least as good as reconstructions using standard inversion algorithms for ISM data, but our method follows a simpler recipe that requires no mathematical insight. Finally, we apply RL deconvolution to merge a series of ten images with varying signal and resolution levels. This combination is relevant to gated stimulated-emission depletion (STED) microscopy, and shows that merges of high-quality images are possible even in cases for which a non-iterative inversion algorithm is unknown. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Angularly-selective transmission imaging in a scanning electron microscope.

PubMed

Holm, Jason; Keller, Robert R

2016-08-01

This work presents recent advances in transmission scanning electron microscopy (t-SEM) imaging control capabilities. A modular aperture system and a cantilever-style sample holder that enable comprehensive angular selectivity of forward-scattered electrons are described. When combined with a commercially available solid-state transmission detector having only basic bright-field and dark-field imaging capabilities, the advances described here enable numerous transmission imaging modes. Several examples are provided that demonstrate how contrast arising from diffraction to mass-thickness can be obtained. Unanticipated image contrast at some imaging conditions is also observed and addressed. Published by Elsevier B.V.

Optimal lens design and use in laser-scanning microscopy

PubMed Central

Negrean, Adrian; Mansvelder, Huibert D.

2014-01-01

In laser-scanning microscopy often an off-the-shelf achromatic doublet is used as a scan lens which can reduce the available diffraction-limited field-of-view (FOV) by a factor of 3 and introduce chromatic aberrations that are scan angle dependent. Here we present several simple lens designs of superior quality that fully make use of high-NA low-magnification objectives, offering diffraction-limited imaging over a large FOV and wavelength range. We constructed a two-photon laser-scanning microscope with optimized custom lenses which had a near diffraction limit point-spread-function (PSF) with less than 3.6% variation over a 400 µm FOV and less than 0.5 µm lateral color between 750 and 1050 nm. PMID:24877017

EDITORIAL: Three decades of scanning tunnelling microscopy that changed the course of surface science Three decades of scanning tunnelling microscopy that changed the course of surface science

NASA Astrophysics Data System (ADS)

Ramachandra Rao, M. S.; Margaritondo, Giorgio

2011-11-01

Three decades ago, with a tiny tip of platinum, the scientific world saw the real space imaging of single atoms with unprecedented spatial resolution. This signalled the birth of one of the most versatile surface probes, based on the physics of quantum mechanical tunnelling: the scanning tunnelling microscope (STM). Invented in 1981 by Gerd Binnig and Heinrich Rohrer of IBM, Zurich, it led to their award of the 1986 Nobel Prize. Atoms, once speculated to be abstract entities used by theoreticians for mere calculations, can be seen to exist for real with the nano-eye of an STM tip that also gives real-space images of molecules and adsorbed complexes on surfaces. From a very fundamental perspective, the STM changed the course of surface science and engineering. STM also emerged as a powerful tool to study various fundamental phenomena relevant to the properties of surfaces in technological applications such as tribology, medical implants, catalysis, sensors and biology—besides elucidating the importance of local bonding geometries and defects, non-periodic structures and the co-existence of nano-scale phases. Atom-level probing, once considered a dream, has seen the light with the evolution of STM. An important off-shoot of STM was the atomic force microscope (AFM) for surface mapping of insulating samples. Then followed the development of a flurry of techniques under the general name of scanning probe microscopy (SPM). These techniques (STM, AFM, MFM, PFM etc) designed for atomic-scale-resolution imaging and spectroscopy, have led to brand new developments in surface analysis. All of these novel methods enabled researchers in recent years to image and analyse complex surfaces on microscopic and nanoscopic scales. All of them utilize a small probe for sensing the surface. The invention of AFM by Gerd Binnig, Calvin Quate and Christopher Gerber opened up new opportunities for characterization of a variety of materials, and various industrial applications could be envisaged. AFM observations of thin-film surfaces give us a picture of surface topography and morphology and any visible defects. The growing importance of ultra-thin films for magnetic recording in hard disk drive systems requires an in-depth understanding of the fundamental mechanisms occurring during growth. This special issue of Journal of Physics D: Applied Physics covers all of the different aspects of SPM that illustrate the achievements of this methodology: nanoscale imaging and mapping (Chiang, and Douillard and Charra), piezoresponse force microscopy (Soergel) and STM engineering (Okuyama and Hamada, and Huang et al). Chiang takes the reader on a journey along the STM imaging of atoms and molecules on surfaces. Jesse and Kalinin explore the band excitations that occur during the corresponding processes. Jia et al propose STM and molecular beam epitaxy as a winning experimental combination at the interface of science and technology. Douillard and Charra describe the high-resolution mapping of plasmonic modes using photoemission and scanning tunnelling microscopy. Cricenti et al demonstrate the importance of SPM in material science and biology. Wiebe et al have probed atomic scale magnetism, revealed by spin polarized scanning tunnelling microscopy. In addition, Simon et al present Fourier transform scanning tunnelling spectroscopy and the possibility to obtain constant energy maps and band dispersion using local measurements. Lackinger and Heckl give a perspective of the use of STM to study covalent intermolecular coupling reactions on surfaces. Okuyama and Hamada investigated hydrogen bond imaging and engineering with STM. Soergel describes the study of substrate-dependent self-assembled CuPc molecules using piezo force microscope (PFM). We are very grateful to the authors and reviewers for the papers in this special issue of Journal of Physics D: Applied Physics. Their contributions have provided a comprehensive picture of the evolution, status and potential of scanning probe microscopy, conveying to the readers the full excitement of this forefront domain of physics.

Note: Fabrication and characterization of molybdenum tips for scanning tunneling microscopy and spectroscopy

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

Carrozzo, P.; Tumino, F.; Facibeni, A.

We present a method for the preparation of bulk molybdenum tips for Scanning Tunneling Microscopy and Spectroscopy and we assess their potential in performing high resolution imaging and local spectroscopy by measurements on different single crystal surfaces in UHV, namely, Au(111), Si(111)-7 × 7, and titanium oxide 2D ordered nanostructures supported on Au(111). The fabrication method is versatile and can be extended to other metals, e.g., cobalt.

Big, Deep, and Smart Data in Scanning Probe Microscopy.

PubMed

Kalinin, Sergei V; Strelcov, Evgheni; Belianinov, Alex; Somnath, Suhas; Vasudevan, Rama K; Lingerfelt, Eric J; Archibald, Richard K; Chen, Chaomei; Proksch, Roger; Laanait, Nouamane; Jesse, Stephen

2016-09-27

Scanning probe microscopy (SPM) techniques have opened the door to nanoscience and nanotechnology by enabling imaging and manipulation of the structure and functionality of matter at nanometer and atomic scales. Here, we analyze the scientific discovery process in SPM by following the information flow from the tip-surface junction, to knowledge adoption by the wider scientific community. We further discuss the challenges and opportunities offered by merging SPM with advanced data mining, visual analytics, and knowledge discovery technologies.

Development of first ever scanning probe microscopy capabilities for plutonium

NASA Astrophysics Data System (ADS)

Beaux, Miles F.; Cordoba, Miguel Santiago; Zocco, Adam T.; Vodnik, Douglas R.; Ramos, Michael; Richmond, Scott; Moore, David P.; Venhaus, Thomas J.; Joyce, Stephen A.; Usov, Igor O.

2017-04-01

Scanning probe microscopy capabilities have been developed for plutonium and its derivative compounds. Specifically, a scanning tunneling microscope and an atomic force microscope housed in an ultra-high vacuum system and an inert atmosphere glove box, respectively, were prepared for the introduction of small non-dispersible δ-Pu coupons. Experimental details, procedures, and preliminary imaging of δ-Pu coupons are presented to demonstrate the functionality of these new capabilities. These first of a kind capabilities for plutonium represent a significant step forward in the ability to characterize and understand plutonium surfaces with high spatial resolution.

Developments in Scanning Hall Probe Microscopy

NASA Astrophysics Data System (ADS)

Chouinard, Taras; Chu, Ricky; David, Nigel; Broun, David

2009-05-01

Low temperature scanning Hall probe microscopy is a sensitive means of imaging magnetic structures with high spatial resolution and magnetic flux sensitivity approaching that of a Superconducting Quantum Interference Device. We have developed a scanning Hall probe microscope with novel features, including highly reliable coarse positioning, in situ optimization of sensor-sample alignment and capacitive transducers for linear, long range positioning measurement. This has been motivated by the need to reposition accurately above fabricated nanostructures such as small superconducting rings. Details of the design and performance will be presented as well as recent progress towards time-resolved measurements with sub nanosecond resolution.

Development of first ever scanning probe microscopy capabilities for plutonium

DOE PAGES

Beaux, Miles F.; Cordoba, Miguel Santiago; Zocco, Adam T.; ...

2017-04-01

Scanning probe microscopy capabilities have been developed for plutonium and its derivative compounds. Specifically, a scanning tunneling microscope and an atomic force microscope housed in an ultra-high vacuum system and an inert atmosphere glove box, respectively, were prepared for the introduction of small non-dispersible δ-Pu coupons. Experimental details, procedures, and preliminary imaging of δ-Pu coupons are presented to demonstrate the functionality of these new capabilities. In conclusion, these first of a kind capabilities for plutonium represent a significant step forward in the ability to characterize and understand plutonium surfaces with high spatial resolution.

Revolving scanning transmission electron microscopy: correcting sample drift distortion without prior knowledge.

PubMed

Sang, Xiahan; LeBeau, James M

2014-03-01

We report the development of revolving scanning transmission electron microscopy--RevSTEM--a technique that enables characterization and removal of sample drift distortion from atomic resolution images without the need for a priori crystal structure information. To measure and correct the distortion, we acquire an image series while rotating the scan coordinate system between successive frames. Through theory and experiment, we show that the revolving image series captures the information necessary to analyze sample drift rate and direction. At atomic resolution, we quantify the image distortion using the projective standard deviation, a rapid, real-space method to directly measure lattice vector angles. By fitting these angles to a physical model, we show that the refined drift parameters provide the input needed to correct distortion across the series. We demonstrate that RevSTEM simultaneously removes the need for a priori structure information to correct distortion, leads to a dramatically improved signal-to-noise ratio, and enables picometer precision and accuracy regardless of drift rate. Copyright © 2013 Elsevier B.V. All rights reserved.

Transmission environmental scanning electron microscope with scintillation gaseous detection device.

PubMed

Danilatos, Gerasimos; Kollia, Mary; Dracopoulos, Vassileios

2015-03-01

A transmission environmental scanning electron microscope with use of a scintillation gaseous detection device has been implemented. This corresponds to a transmission scanning electron microscope but with addition of a gaseous environment acting both as environmental and detection medium. A commercial type of low vacuum machine has been employed together with appropriate modifications to the detection configuration. This involves controlled screening of various emitted signals in conjunction with a scintillation gaseous detection device already provided with the machine for regular surface imaging. Dark field and bright field imaging has been obtained along with other detection conditions. With a progressive series of modifications and tests, the theory and practice of a novel type of microscopy is briefly shown now ushering further significant improvements and developments in electron microscopy as a whole. Copyright © 2014 Elsevier B.V. All rights reserved.

Experiments on terahertz 3D scanning microscopic imaging

NASA Astrophysics Data System (ADS)

Zhou, Yi; Li, Qi

2016-10-01

Compared with the visible light and infrared, terahertz (THz) radiation can penetrate nonpolar and nonmetallic materials. There are many studies on the THz coaxial transmission confocal microscopy currently. But few researches on the THz dual-axis reflective confocal microscopy were reported. In this paper, we utilized a dual-axis reflective confocal scanning microscope working at 2.52 THz. In contrast with the THz coaxial transmission confocal microscope, the microscope adopted in this paper can attain higher axial resolution at the expense of reduced lateral resolution, revealing more satisfying 3D imaging capability. Objects such as Chinese characters "Zhong-Hua" written in paper with a pencil and a combined sheet metal which has three layers were scanned. The experimental results indicate that the system can extract two Chinese characters "Zhong," "Hua" or three layers of the combined sheet metal. It can be predicted that the microscope can be applied to biology, medicine and other fields in the future due to its favorable 3D imaging capability.

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.

Video-rate hyperspectral two-photon fluorescence microscopy for in vivo imaging

NASA Astrophysics Data System (ADS)

Deng, Fengyuan; Ding, Changqin; Martin, Jerald C.; Scarborough, Nicole M.; Song, Zhengtian; Eakins, Gregory S.; Simpson, Garth J.

2018-02-01

Fluorescence hyperspectral imaging is a powerful tool for in vivo biological studies. The ability to recover the full spectra of the fluorophores allows accurate classification of different structures and study of the dynamic behaviors during various biological processes. However, most existing methods require significant instrument modifications and/or suffer from image acquisition rates too low for compatibility with in vivo imaging. In the present work, a fast (up to 18 frames per second) hyperspectral two-photon fluorescence microscopy approach was demonstrated. Utilizing the beamscanning hardware inherent in conventional multi-photon microscopy, the angle dependence of the generated fluorescence signal as a function beam's position allowed the system to probe of a different potion of the spectrum at every single scanning line. An iterative algorithm to classify the fluorophores recovered spectra with up to 2,400 channels using a custom high-speed 16-channel photon multiplier tube array. Several dynamic samples including live fluorescent labeled C. elegans were imaged at video rate. Fluorescence spectra recovered using no a priori spectral information agreed well with those obtained by fluorimetry. This system required minimal changes to most existing beam-scanning multi-photon fluorescence microscopes, already accessible in many research facilities.

Enhanced light element imaging in atomic resolution scanning transmission electron microscopy.

PubMed

Findlay, S D; Kohno, Y; Cardamone, L A; Ikuhara, Y; Shibata, N

2014-01-01

We show that an imaging mode based on taking the difference between signals recorded from the bright field (forward scattering region) in atomic resolution scanning transmission electron microscopy provides an enhancement of the detectability of light elements over existing techniques. In some instances this is an enhancement of the visibility of the light element columns relative to heavy element columns. In all cases explored it is an enhancement in the signal-to-noise ratio of the image at the light column site. The image formation mechanisms are explained and the technique is compared with earlier approaches. Experimental data, supported by simulation, are presented for imaging the oxygen columns in LaAlO₃. Case studies looking at imaging hydrogen columns in YH₂ and lithium columns in Al₃Li are also explored through simulation, particularly with respect to the dependence on defocus, probe-forming aperture angle and detector collection aperture angles. © 2013 Elsevier B.V. All rights reserved.

Field emission scanning electron microscopy (FE-SEM) as an approach for nanoparticle detection inside cells.

PubMed

Havrdova, M; Polakova, K; Skopalik, J; Vujtek, M; Mokdad, A; Homolkova, M; Tucek, J; Nebesarova, J; Zboril, R

2014-12-01

When developing new nanoparticles for bio-applications, it is important to fully characterize the nanoparticle's behavior in biological systems. The most common techniques employed for mapping nanoparticles inside cells include transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). These techniques entail passing an electron beam through a thin specimen. STEM or TEM imaging is often used for the detection of nanoparticles inside cellular organelles. However, lengthy sample preparation is required (i.e., fixation, dehydration, drying, resin embedding, and cutting). In the present work, a new matrix (FTO glass) for biological samples was used and characterized by field emission scanning electron microscopy (FE-SEM) to generate images comparable to those obtained by TEM. Using FE-SEM, nanoparticle images were acquired inside endo/lysosomes without disruption of the cellular shape. Furthermore, the initial steps of nanoparticle incorporation into the cells were captured. In addition, the conductive FTO glass endowed the sample with high stability under the required accelerating voltage. Owing to these features of the sample, further analyses could be performed (material contrast and energy-dispersive X-ray spectroscopy (EDS)), which confirmed the presence of nanoparticles inside the cells. The results showed that FE-SEM can enable detailed characterization of nanoparticles in endosomes without the need for contrast staining or metal coating of the sample. Images showing the intracellular distribution of nanoparticles together with cellular morphology can give important information on the biocompatibility and demonstrate the potential of nanoparticle utilization in medicine. Copyright © 2014 Elsevier Ltd. All rights reserved.

Denoising time-resolved microscopy image sequences with singular value thresholding.

PubMed

Furnival, Tom; Leary, Rowan K; Midgley, Paul A

2017-07-01

Time-resolved imaging in microscopy is important for the direct observation of a range of dynamic processes in both the physical and life sciences. However, the image sequences are often corrupted by noise, either as a result of high frame rates or a need to limit the radiation dose received by the sample. Here we exploit both spatial and temporal correlations using low-rank matrix recovery methods to denoise microscopy image sequences. We also make use of an unbiased risk estimator to address the issue of how much thresholding to apply in a robust and automated manner. The performance of the technique is demonstrated using simulated image sequences, as well as experimental scanning transmission electron microscopy data, where surface adatom motion and nanoparticle structural dynamics are recovered at rates of up to 32 frames per second. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Tackling the Challenges of Dynamic Experiments Using Liquid-Cell Transmission Electron Microscopy.

PubMed

Parent, Lucas R; Bakalis, Evangelos; Proetto, Maria; Li, Yiwen; Park, Chiwoo; Zerbetto, Francesco; Gianneschi, Nathan C

2018-01-16

Revolutions in science and engineering frequently result from the development, and wide adoption, of a new, powerful characterization or imaging technique. Beginning with the first glass lenses and telescopes in astronomy, to the development of visual-light microscopy, staining techniques, confocal microscopy, and fluorescence super-resolution microscopy in biology, and most recently aberration-corrected, cryogenic, and ultrafast (4D) electron microscopy, X-ray microscopy, and scanning probe microscopy in nanoscience. Through these developments, our perception and understanding of the physical nature of matter at length-scales beyond ordinary perception have been fundamentally transformed. Despite this progression in microscopy, techniques for observing nanoscale chemical processes and solvated/hydrated systems are limited, as the necessary spatial and temporal resolution presents significant technical challenges. However, the standard reliance on indirect or bulk phase characterization of nanoscale samples in liquids is undergoing a shift in recent times with the realization ( Williamson et al. Nat. Mater . 2003 , 2 , 532 - 536 ) of liquid-cell (scanning) transmission electron microscopy, LC(S)TEM, where picoliters of solution are hermetically sealed between electron-transparent "windows," which can be directly imaged or videoed at the nanoscale using conventional transmission electron microscopes. This Account seeks to open a discussion on the topic of standardizing strategies for conducting imaging experiments with a view to characterizing dynamics and motion of nanoscale materials. This is a challenge that could be described by critics and proponents alike, as analogous to doing chemistry in a lightning storm; where the nature of the solution, the nanomaterial, and the dynamic behaviors are all potentially subject to artifactual influence by the very act of our observation.

Silicon technology-based micro-systems for atomic force microscopy/photon scanning tunnelling microscopy.

PubMed

Gall-Borrut, P; Belier, B; Falgayrettes, P; Castagne, M; Bergaud, C; Temple-Boyer, P

2001-04-01

We developed silicon nitride cantilevers integrating a probe tip and a wave guide that is prolonged on the silicon holder with one or two guides. A micro-system is bonded to a photodetector. The resulting hybrid system enables us to obtain simultaneously topographic and optical near-field images. Examples of images obtained on a longitudinal cross-section of an optical fibre are shown.

Correlative atomic force microscopy quantitative imaging-laser scanning confocal microscopy quantifies the impact of stressors on live cells in real-time.

PubMed

Bhat, Supriya V; Sultana, Taranum; Körnig, André; McGrath, Seamus; Shahina, Zinnat; Dahms, Tanya E S

2018-05-29

There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.

Quantitative analysis of the effect of environmental-scanning electron microscopy on collagenous tissues.

PubMed

Lee, Woowon; Toussaint, Kimani C

2018-05-31

Environmental-scanning electron microscopy (ESEM) is routinely applied to various biological samples due to its ability to maintain a wet environment while imaging; moreover, the technique obviates the need for sample coating. However, there is limited research carried out on electron-beam (e-beam) induced tissue damage resulting from using the ESEM. In this paper, we use quantitative second-harmonic generation (SHG) microscopy to examine the effects of e-beam exposure from the ESEM on collagenous tissue samples prepared as either fixed, frozen, wet or dehydrated. Quantitative SHG analysis of tissues, before and after ESEM e-beam exposure in low-vacuum mode, reveals evidence of cross-linking of collagen fibers, however there are no structural differences observed in fixed tissue. Meanwhile wet-mode ESEM appears to radically alter the structure from a regular fibrous arrangement to a more random fiber orientation. We also confirm that ESEM images of collagenous tissues show higher spatial resolution compared to SHG microscopy, but the relative tradeoff with collagen specificity reduces its effectiveness in quantifying collagen fiber organization. Our work provides insight on both the limitations of the ESEM for tissue imaging, and the potential opportunity to use as a complementary technique when imaging fine features in the non-collagenous regions of tissue samples.

Combined Multidimensional Microscopy as a Histopathology Imaging Tool.

PubMed

Shami, Gerald J; Cheng, Delfine; Braet, Filip

2017-02-01

Herein, we present a highly versatile bioimaging workflow for the multidimensional imaging of biological structures across vastly different length scales. Such an approach allows for the optimised preparation of samples in one go for consecutive X-ray micro-computed tomography, bright-field light microscopy and backscattered scanning electron microscopy, thus, facilitating the disclosure of combined structural information ranging from the gross tissue or cellular level, down to the nanometre scale. In this current study, we characterize various aspects of the hepatic vasculature, ranging from such large vessels as branches of the hepatic portal vein and hepatic artery, down to the smallest sinusoidal capillaries. By employing high-resolution backscattered scanning electron microscopy, we were able to further characterize the subcellular features of a range of hepatic sinusoidal cells including, liver sinusoidal endothelial cells, pit cells and Kupffer cells. Above all, we demonstrate the capabilities of a specimen manipulation workflow that can be applied and adapted to a plethora of functional and structural investigations and experimental models. Such an approach harnesses the fundamental advantages inherent to the various imaging modalities presented herein, and when combined, offers information not currently available by any single imaging platform. J. Cell. Physiol. 232: 249-256, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Parallelized multi–graphics processing unit framework for high-speed Gabor-domain optical coherence microscopy

PubMed Central

Tankam, Patrice; Santhanam, Anand P.; Lee, Kye-Sung; Won, Jungeun; Canavesi, Cristina; Rolland, Jannick P.

2014-01-01

Abstract. Gabor-domain optical coherence microscopy (GD-OCM) is a volumetric high-resolution technique capable of acquiring three-dimensional (3-D) skin images with histological resolution. Real-time image processing is needed to enable GD-OCM imaging in a clinical setting. We present a parallelized and scalable multi-graphics processing unit (GPU) computing framework for real-time GD-OCM image processing. A parallelized control mechanism was developed to individually assign computation tasks to each of the GPUs. For each GPU, the optimal number of amplitude-scans (A-scans) to be processed in parallel was selected to maximize GPU memory usage and core throughput. We investigated five computing architectures for computational speed-up in processing 1000×1000 A-scans. The proposed parallelized multi-GPU computing framework enables processing at a computational speed faster than the GD-OCM image acquisition, thereby facilitating high-speed GD-OCM imaging in a clinical setting. Using two parallelized GPUs, the image processing of a 1×1×0.6  mm3 skin sample was performed in about 13 s, and the performance was benchmarked at 6.5 s with four GPUs. This work thus demonstrates that 3-D GD-OCM data may be displayed in real-time to the examiner using parallelized GPU processing. PMID:24695868

Parallelized multi-graphics processing unit framework for high-speed Gabor-domain optical coherence microscopy.

PubMed

Tankam, Patrice; Santhanam, Anand P; Lee, Kye-Sung; Won, Jungeun; Canavesi, Cristina; Rolland, Jannick P

2014-07-01

Gabor-domain optical coherence microscopy (GD-OCM) is a volumetric high-resolution technique capable of acquiring three-dimensional (3-D) skin images with histological resolution. Real-time image processing is needed to enable GD-OCM imaging in a clinical setting. We present a parallelized and scalable multi-graphics processing unit (GPU) computing framework for real-time GD-OCM image processing. A parallelized control mechanism was developed to individually assign computation tasks to each of the GPUs. For each GPU, the optimal number of amplitude-scans (A-scans) to be processed in parallel was selected to maximize GPU memory usage and core throughput. We investigated five computing architectures for computational speed-up in processing 1000×1000 A-scans. The proposed parallelized multi-GPU computing framework enables processing at a computational speed faster than the GD-OCM image acquisition, thereby facilitating high-speed GD-OCM imaging in a clinical setting. Using two parallelized GPUs, the image processing of a 1×1×0.6  mm3 skin sample was performed in about 13 s, and the performance was benchmarked at 6.5 s with four GPUs. This work thus demonstrates that 3-D GD-OCM data may be displayed in real-time to the examiner using parallelized GPU processing.

Confocal raman microscopy as a non-invasive tool to investigate the phase composition of frozen complex cryopreservation media.

PubMed

Kreiner-Møller, A; Stracke, F; Zimmermann, H

2013-01-01

Various cryoprotective agents (CPA) are added to cell media in order to avoid cell injury during cryo preservation. The resulting complex environment of the preserved cell, consisting of crystalline and liquid phases can however not be investigated non-invasively by established methods in cryobiology. This study shows how scanning confocal Raman microscopy can non-invasively extract information on chemical composition, phase domain and distribution at cryogenic temperatures. The formation of the salt hydrate, hydrohalite NaCl∙H2O, in solutions comprised of phosphate buffered saline (PBS) and dimethyl sulphoxide (DMSO) is studied in particular. Scanning confocal Raman microscopy can be used to unambiguously identify hydrohalite in a medium containing DMSO and saline. The confocal Raman microscopy imaging along with differential scanning calorimetric measurements further show that the hydrohalite is formed without eutectic formation. This method also allows for discrimination between closely packed hydrohalite crystals that are oriented differently.

Simultaneous Scanning Ion Conductance Microscopy and Atomic Force Microscopy with Microchanneled Cantilevers

NASA Astrophysics Data System (ADS)

Ossola, Dario; Dorwling-Carter, Livie; Dermutz, Harald; Behr, Pascal; Vörös, János; Zambelli, Tomaso

2015-12-01

We combined scanning ion conductance microscopy (SICM) and atomic force microscopy (AFM) into a single tool using AFM cantilevers with an embedded microchannel flowing into the nanosized aperture at the apex of the hollow pyramid. An electrode was positioned in the AFM fluidic circuit connected to a second electrode in the bath. We could thus simultaneously measure the ionic current and the cantilever bending (in optical beam deflection mode). First, we quantitatively compared the SICM and AFM contact points on the approach curves. Second, we estimated where the probe in SICM mode touches the sample during scanning on a calibration grid and applied the finding to image a network of neurites on a Petri dish. Finally, we assessed the feasibility of a double controller using both the ionic current and the deflection as input signals of the piezofeedback. The experimental data were rationalized in the framework of finite elements simulations.

Cytocompatibility and uptake of halloysite clay nanotubes.

PubMed

Vergaro, Viviana; Abdullayev, Elshad; Lvov, Yuri M; Zeitoun, Andre; Cingolani, Roberto; Rinaldi, Ross; Leporatti, Stefano

2010-03-08

Halloysite is aluminosilicate clay with hollow tubular structure of 50 nm external diameter and 15 nm diameter lumen. Halloysite biocompatibility study is important for its potential applications in polymer composites, bone implants, controlled drug delivery, and for protective coating (e.g., anticorrosion or antimolding). Halloysite nanotubes were added to different cell cultures for toxicity tests. Its fluorescence functionalization by aminopropyltriethosilane (APTES) and with fluorescently labeled polyelectrolyte layers allowed following halloysite uptake by the cells with confocal laser scanning microscopy (CLSM). Quantitative Trypan blue and MTT measurements performed with two neoplastic cell lines model systems as a function of the nanotubes concentration and incubation time indicate that halloysite exhibits a high level of biocompatibility and very low cytotoxicity, rendering it a good candidate for household materials and medicine. A combination of transmission electron microscopy (TEM), scanning electron microscopy (SEM), and scanning force microscopy (SFM) imaging techniques have been employed to elucidate the structure of halloysite nanotubes.

Imaging of surface spin textures on bulk crystals by scanning electron microscopy

NASA Astrophysics Data System (ADS)

Akamine, Hiroshi; Okumura, So; Farjami, Sahar; Murakami, Yasukazu; Nishida, Minoru

2016-11-01

Direct observation of magnetic microstructures is vital for advancing spintronics and other technologies. Here we report a method for imaging surface domain structures on bulk samples by scanning electron microscopy (SEM). Complex magnetic domains, referred to as the maze state in CoPt/FePt alloys, were observed at a spatial resolution of less than 100 nm by using an in-lens annular detector. The method allows for imaging almost all the domain walls in the mazy structure, whereas the visualisation of the domain walls with the classical SEM method was limited. Our method provides a simple way to analyse surface domain structures in the bulk state that can be used in combination with SEM functions such as orientation or composition analysis. Thus, the method extends applications of SEM-based magnetic imaging, and is promising for resolving various problems at the forefront of fields including physics, magnetics, materials science, engineering, and chemistry.

Signal and noise modeling in confocal laser scanning fluorescence microscopy.

PubMed

Herberich, Gerlind; Windoffer, Reinhard; Leube, Rudolf E; Aach, Til

2012-01-01

Fluorescence confocal laser scanning microscopy (CLSM) has revolutionized imaging of subcellular structures in biomedical research by enabling the acquisition of 3D time-series of fluorescently-tagged proteins in living cells, hence forming the basis for an automated quantification of their morphological and dynamic characteristics. Due to the inherently weak fluorescence, CLSM images exhibit a low SNR. We present a novel model for the transfer of signal and noise in CLSM that is both theoretically sound as well as corroborated by a rigorous analysis of the pixel intensity statistics via measurement of the 3D noise power spectra, signal-dependence and distribution. Our model provides a better fit to the data than previously proposed models. Further, it forms the basis for (i) the simulation of the CLSM imaging process indispensable for the quantitative evaluation of CLSM image analysis algorithms, (ii) the application of Poisson denoising algorithms and (iii) the reconstruction of the fluorescence signal.

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

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

Deng, Junjing; Vine, David J.; Chen, Si

X-ray microscopy can be used to image whole, unsectioned cells in their native hydrated state. It complements the higher resolution of electron microscopy for submicrometer thick specimens, and the molecule-specific imaging capabilites of fluorescence light microscopy. We describe here the first use of fast, continuous x-ray scanning of frozen hydrated cells for simultaneous sub-20 nm resolution ptychographic transmission imaging with high contrast, and sub-100 nm resolution deconvolved x-ray fluorescence imaging of diffusible and bound ions at native concentrations, without the need to add specific labels. Here, by working with cells that have been rapidly frozen without the use of chemicalmore » fixatives, and imaging them under cryogenic conditions, we are able to obtain images with well preserved structural and chemical composition, and sufficient stability against radiation damage to allow for multiple images to be obtained with no observable change.« less

Imaging quantum transport using scanning gate microscopy

NASA Astrophysics Data System (ADS)

Hackens, Benoit

2014-03-01

Quantum transport in nanodevices is usually probed thanks to measurements of the electrical resistance or conductance, which lack the spatial resolution necessary to probe electron behaviour inside the devices. In this talk, we will show that scanning gate microscopy (SGM) yields real-space images of quantum transport phenomena inside archetypal mesoscopic devices such as quantum point contacts and quantum rings. We will first discuss the SGM technique, which is based on mapping the electrical conductance of a device as an electrically-biased sharp metallic tip scans in its vicinity. With SGM, we demonstrated low temperature imaging of the electron probability density and interferences in embedded mesoscopic quantum rings [B. Hackens et al., Nat. Phys. 2, 826 (2006)]. At high magnetic field, thanks to the SGM conductance maps, one can decrypt complex transport phenomena such as tunneling between quantum Hall edge state, either direct or through localized states [B. Hackens et al., Nat. Comm. 1, 39 (2010)]. Moreover, the technique also allows to perform local spectroscopy of electron transport through selected localized states [F. Martins et al., New J. of Phys. 15, 013049 (2013); F. Martins et al., Sci. Rep. 3, 1416 (2013)]. Overall, these examples show that scanning gate microscopy is a powerful tool for imaging charge carrier behavior inside devices fabricated from a variety of materials, and opens the way towards a more intimate manipulation of charge and quasiparticle transport. This work was performed in collaboration with F. Martins, S. Faniel, B. Brun, M. Pala, X. Wallart, L. Desplanque, B. Rosenow, T. Ouisse, H. Sellier, S. Huant and V. Bayot.

Scanning gate microscopy of quantum rings: effects of an external magnetic field and of charged defects.

PubMed

Pala, M G; Baltazar, S; Martins, F; Hackens, B; Sellier, H; Ouisse, T; Bayot, V; Huant, S

2009-07-01

We study scanning gate microscopy (SGM) in open quantum rings obtained from buried semiconductor InGaAs/InAlAs heterostructures. By performing a theoretical analysis based on the Keldysh-Green function approach we interpret the radial fringes observed in experiments as the effect of randomly distributed charged defects. We associate SGM conductance images with the local density of states (LDOS) of the system. We show that such an association cannot be made with the current density distribution. By varying an external magnetic field we are able to reproduce recursive quasi-classical orbits in LDOS and conductance images, which bear the same periodicity as the Aharonov-Bohm effect.

Recent advancements in nanoelectrodes and nanopipettes used in combined scanning electrochemical microscopy techniques.

PubMed

Kranz, Christine

2014-01-21

In recent years, major developments in scanning electrochemical microscopy (SECM) have significantly broadened the application range of this electroanalytical technique from high-resolution electrochemical imaging via nanoscale probes to large scale mapping using arrays of microelectrodes. A major driving force in advancing the SECM methodology is based on developing more sophisticated probes beyond conventional micro-disc electrodes usually based on noble metals or carbon microwires. This critical review focuses on the design and development of advanced electrochemical probes particularly enabling combinations of SECM with other analytical measurement techniques to provide information beyond exclusively measuring electrochemical sample properties. Consequently, this critical review will focus on recent progress and new developments towards multifunctional imaging.

Imaging three-dimensional light propagation through periodic nanohole arrays using scanning aperture microscopy

PubMed Central

Chowdhury, Mustafa H.; Catchmark, Jeffrey M.; Lakowicz, Joseph R.

2009-01-01

The authors introduce a technique for three-dimensional (3D) imaging of the light transmitted through periodic nanoapertures using a scanning probe to perform optical sectioning microscopy. For a 4×4 nanohole array, the transmitted light displays intensity modulations along the propagation axis, with the maximum intensity occurring at 450 μm above the surface. The propagating fields show low divergence, suggesting a beaming effect induced by the array. At distances within 25 μm from the surface, they observe subwavelength confinement of light propagating from the individual nanoholes. Hence, this technique can potentially be used to map the 3D distribution of propagating light, with high spatial resolution. PMID:19696912

Atomic bonding effects in annular dark field scanning transmission electron microscopy. II. Experiments

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

Odlyzko, Michael L.; Held, Jacob T.; Mkhoyan, K. Andre, E-mail: mkhoyan@umn.edu

2016-07-15

Quantitatively calibrated annular dark field scanning transmission electron microscopy (ADF-STEM) imaging experiments were compared to frozen phonon multislice simulations adapted to include chemical bonding effects. Having carefully matched simulation parameters to experimental conditions, a depth-dependent bonding effect was observed for high-angle ADF-STEM imaging of aluminum nitride. This result is explained by computational predictions, systematically examined in the preceding portion of this study, showing the propagation of the converged STEM beam to be highly sensitive to net interatomic charge transfer. Thus, although uncertainties in experimental conditions and simulation accuracy remain, the computationally predicted experimental bonding effect withstands the experimental testing reportedmore » here.« less

Nanometer-scale surface potential and resistance mapping of wide-bandgap Cu(In,Ga)Se2 thin films

NASA Astrophysics Data System (ADS)

Jiang, C.-S.; Contreras, M. A.; Mansfield, L. M.; Moutinho, H. R.; Egaas, B.; Ramanathan, K.; Al-Jassim, M. M.

2015-01-01

We report microscopic characterization studies of wide-bandgap Cu(In,Ga)Se2 photovoltaic thin films using the nano-electrical probes of scanning Kelvin probe force microscopy and scanning spreading resistance microscopy. With increasing bandgap, the potential imaging shows significant increases in both the large potential features due to extended defects or defect aggregations and the potential fluctuation due to unresolvable point defects with single or a few charges. The resistance imaging shows increases in both overall resistance and resistance nonuniformity due to defects in the subsurface region. These defects are expected to affect open-circuit voltage after the surfaces are turned to junction upon device completion.

Magnetic elements for switching magnetization magnetic force microscopy tips.

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

Cambel, V.; Elias, P.; Gregusova, D.

2010-09-01

Using combination of micromagnetic calculations and magnetic force microscopy (MFM) imaging we find optimal parameters for novel magnetic tips suitable for switching magnetization MFM. Switching magnetization MFM is based on two-pass scanning atomic force microscopy with reversed tip magnetization between the scans. Within the technique the sum of the scanned data with reversed tip magnetization depicts local atomic forces, while their difference maps the local magnetic forces. Here we propose the design and calculate the magnetic properties of tips suitable for this scanning probe technique. We find that for best performance the spin-polarized tips must exhibit low magnetic moment, lowmore » switching fields, and single-domain state at remanence. The switching field of such tips is calculated and optimum shape of the Permalloy elements for the tips is found. We show excellent correspondence between calculated and experimental results for Py elements.« less

The application of scanning electron microscopy to fractography

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

Brooks, C.R.; McGill, B.L.

1994-10-01

Many failures involve fracture, and determination of the fracture process is a key factor in understanding the failure. This is frequently accomplished by characterizing the topography of the fracture surface. Scanning electron microscopy has a prominent role in fractography due to three features of the scanning electron microscope (SEM): high resolution, great depth of field, and the ability to obtain chemical information via analysis of the X-rays generated by the electrons. A qualitative treatment is presented of the interaction of electrons with a sample and the effect of the SEM operating parameters on image formation, quality, and X-ray analysis. Fractographsmore » are presented to illustrate these features of scanning electron microscopy and to illustrate the limitations and precautions in obtaining fractographs and x-ray analyses. The review is concluded with examples of fracture surface features of metallic, ceramic, and polymeric materials.« less

Periodic scarred States in open quantum dots as evidence of quantum Darwinism.

PubMed

Burke, A M; Akis, R; Day, T E; Speyer, Gil; Ferry, D K; Bennett, B R

2010-04-30

Scanning gate microscopy (SGM) is used to image scar structures in an open quantum dot, which is created in an InAs quantum well by electron-beam lithography and wet etching. The scanned images demonstrate periodicities in magnetic field that correlate to those found in the conductance fluctuations. Simulations have shown that these magnetic transform images bear a strong resemblance to actual scars found in the dot that replicate through the modes in direct agreement with quantum Darwinism.

Periodic Scarred States in Open Quantum Dots as Evidence of Quantum Darwinism

NASA Astrophysics Data System (ADS)

Burke, A. M.; Akis, R.; Day, T. E.; Speyer, Gil; Ferry, D. K.; Bennett, B. R.

2010-04-01

Scanning gate microscopy (SGM) is used to image scar structures in an open quantum dot, which is created in an InAs quantum well by electron-beam lithography and wet etching. The scanned images demonstrate periodicities in magnetic field that correlate to those found in the conductance fluctuations. Simulations have shown that these magnetic transform images bear a strong resemblance to actual scars found in the dot that replicate through the modes in direct agreement with quantum Darwinism.

Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy.

PubMed

Panier, Thomas; Romano, Sebastián A; Olive, Raphaël; Pietri, Thomas; Sumbre, Germán; Candelier, Raphaël; Debrégeas, Georges

2013-01-01

The optical transparency and the small dimensions of zebrafish at the larval stage make it a vertebrate model of choice for brain-wide in-vivo functional imaging. However, current point-scanning imaging techniques, such as two-photon or confocal microscopy, impose a strong limit on acquisition speed which in turn sets the number of neurons that can be simultaneously recorded. At 5 Hz, this number is of the order of one thousand, i.e., approximately 1-2% of the brain. Here we demonstrate that this limitation can be greatly overcome by using Selective-plane Illumination Microscopy (SPIM). Zebrafish larvae expressing the genetically encoded calcium indicator GCaMP3 were illuminated with a scanned laser sheet and imaged with a camera whose optical axis was oriented orthogonally to the illumination plane. This optical sectioning approach was shown to permit functional imaging of a very large fraction of the brain volume of 5-9-day-old larvae with single- or near single-cell resolution. The spontaneous activity of up to 5,000 neurons was recorded at 20 Hz for 20-60 min. By rapidly scanning the specimen in the axial direction, the activity of 25,000 individual neurons from 5 different z-planes (approximately 30% of the entire brain) could be simultaneously monitored at 4 Hz. Compared to point-scanning techniques, this imaging strategy thus yields a ≃20-fold increase in data throughput (number of recorded neurons times acquisition rate) without compromising the signal-to-noise ratio (SNR). The extended field of view offered by the SPIM method allowed us to directly identify large scale ensembles of neurons, spanning several brain regions, that displayed correlated activity and were thus likely to participate in common neural processes. The benefits and limitations of SPIM for functional imaging in zebrafish as well as future developments are briefly discussed.

Acquisition of multiple image stacks with a confocal laser scanning microscope

NASA Astrophysics Data System (ADS)

Zuschratter, Werner; Steffen, Thomas; Braun, Katharina; Herzog, Andreas; Michaelis, Bernd; Scheich, Henning

1998-06-01

Image acquisition at high magnification is inevitably correlated with a limited view over the entire tissue section. To overcome this limitation we designed software for multiple image-stack acquisition (3D-MISA) in confocal laser scanning microscopy (CLSM). The system consists of a 4 channel Leica CLSM equipped with a high resolution z- scanning stage mounted on a xy-monitorized stage. The 3D- MISA software is implemented into the microscope scanning software and uses the microscope settings for the movements of the xy-stage. It allows storage and recall of 70 xyz- positions and the automatic 3D-scanning of image arrays between selected xyz-coordinates. The number of images within one array is limited only by the amount of disk space or memory available. Although for most applications the accuracy of the xy-scanning stage is sufficient for a precise alignment of tiled views, the software provides the possibility of an adjustable overlap between two image stacks by shifting the moving steps of the xy-scanning stage. After scanning a tiled image gallery of the extended focus-images of each channel will be displayed on a graphic monitor. In addition, a tiled image gallery of individual focal planes can be created. In summary, the 3D-MISA allows 3D-image acquisition of coherent regions in combination with high resolution of single images.

Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

Chemically Resolved Imaging of Biological Cells and Thin Films by Infrared Scanning Near-Field Optical Microscopy

PubMed Central

Cricenti, Antonio; Generosi, Renato; Luce, Marco; Perfetti, Paolo; Margaritondo, Giorgio; Talley, David; Sanghera, Jas S.; Aggarwal, Ishwar D.; Tolk, Norman H.; Congiu-Castellano, Agostina; Rizzo, Mark A.; Piston, David W.

2003-01-01

The infrared (IR) absorption of a biological system can potentially report on fundamentally important microchemical properties. For example, molecular IR profiles are known to change during increases in metabolic flux, protein phosphorylation, or proteolytic cleavage. However, practical implementation of intracellular IR imaging has been problematic because the diffraction limit of conventional infrared microscopy results in low spatial resolution. We have overcome this limitation by using an IR spectroscopic version of scanning near-field optical microscopy (SNOM), in conjunction with a tunable free-electron laser source. The results presented here clearly reveal different chemical constituents in thin films and biological cells. The space distribution of specific chemical species was obtained by taking SNOM images at IR wavelengths (λ) corresponding to stretch absorption bands of common biochemical bonds, such as the amide bond. In our SNOM implementation, this chemical sensitivity is combined with a lateral resolution of 0.1 μm (≈λ/70), well below the diffraction limit of standard infrared microscopy. The potential applications of this approach touch virtually every aspect of the life sciences and medical research, as well as problems in materials science, chemistry, physics, and environmental research. PMID:14507733

Characterization of human breast cancer by scanning acoustic microscopy

NASA Astrophysics Data System (ADS)

Chen, Di; Malyarenko, Eugene; Seviaryn, Fedar; Yuan, Ye; Sherman, Mark; Bandyopadhyay, Sudeshna; Gierach, Gretchen; Greenway, Christopher W.; Maeva, Elena; Strumban, Emil; Duric, Neb; Maev, Roman

2013-03-01

Objectives: The purpose of this study was to characterize human breast cancer tissues by the measurement of microacoustic properties. Methods: We investigated eight breast cancer patients using acoustic microscopy. For each patient, seven blocks of tumor tissue were collected from seven different positions around a tumor mass. Frozen sections (10 micrometer, μm) of human breast cancer tissues without staining and fixation were examined in a scanning acoustic microscope with focused transducers at 80 and 200 MHz. Hematoxylin and Eosin (H and E) stained sections from the same frozen breast cancer tissues were imaged by optical microscopy for comparison. Results: The results of acoustic imaging showed that acoustic attenuation and sound speed in cancer cell-rich tissue regions were significantly decreased compared with the surrounding tissue regions, where most components are normal cells/tissues, such as fibroblasts, connective tissue and lymphocytes. Our observation also showed that the ultrasonic properties were influenced by arrangements of cells and tissue patterns. Conclusions: Our data demonstrate that attenuation and sound speed imaging can provide biomechanical information of the tumor and normal tissues. The results also demonstrate the potential of acoustic microscopy as an auxiliary method for operative detection and localization of cancer affected regions.

Evaluation of agave fiber delignification by means of microscopy techniques and image analysis.

PubMed

Hernández-Hernández, Hilda M; Chanona-Pérez, Jorge J; Calderón-Domínguez, Georgina; Perea-Flores, María J; Mendoza-Pérez, Jorge A; Vega, Alberto; Ligero, Pablo; Palacios-González, Eduardo; Farrera-Rebollo, Reynold R

2014-10-01

Recently, the use of different types of natural fibers to produce paper and textiles from agave plants has been proposed. Agave atrovirens can be a good source of cellulose and lignin; nevertheless, the microstructural changes that happen during delignification have scarcely been studied. The aim of this work was to study the microstructural changes that occur during the delignification of agave fibers by means of microscopy techniques and image analysis. The fibers of A. atrovirens were obtained from leaves using convective drying, milling, and sieving. Fibers were processed using the Acetosolv pulping method at different concentrations of acetic acid; increasing acid concentration promoted higher levels of delignification, structural damage, and the breakdown of fiber clumps. Delignification followed by spectrometric analysis and microstructural studies were carried out by light, confocal laser scanning and scanning electron microscopy and showed that the delignification process follows three stages: initial, bulk, and residual. Microscopy techniques and image analysis were efficient tools for microstructural characterization during delignification of agave fibers, allowing quantitative evaluation of the process and the development of linear prediction models. The data obtained integrated numerical and microstructural information that could be valuable for the study of pulping of lignocellulosic materials.

Multiple speckle illumination for optical-resolution photoacoustic imaging

NASA Astrophysics Data System (ADS)

Poisson, Florian; Stasio, Nicolino; Moser, Christophe; Psaltis, Demetri; Bossy, Emmanuel

2017-03-01

Optical-resolution photoacoustic microscopy offers exquisite and specific contrast to optical absorption. Conventional approaches generally involves raster scanning a focused spot over the sample. Here, we demonstrate that a full-field illumination approach with multiple speckle illumination can also provide diffraction-limited optical-resolution photoacoustic images. Two different proof-of-concepts are demonstrated with micro-structured test samples. The first approach follows the principle of correlation/ghost imaging,1, 2 and is based on cross-correlating photoacoustic signals under multiple speckle illumination with known speckle patterns measured during a calibration step. The second approach is a speckle scanning microscopy technique, which adapts the technique proposed in fluorescence microscopy by Bertolotti and al.:3 in our work, spatially unresolved photoacoustic measurements are performed for various translations of unknown speckle patterns. A phase-retrieval algorithm is used to reconstruct the object from the knowledge of the modulus of its Fourier Transform yielded by the measurements. Because speckle patterns naturally appear in many various situations, including propagation through biological tissue or multi-mode fibers (for which focusing light is either very demanding if not impossible), speckle-illumination-based photoacoustic microscopy provides a powerful framework for the development of novel reconstruction approaches, well-suited to compressed sensing approaches.2

Spontaneous Polarization in Bio-organic Materials Studied by Scanning Pyroelectric Microscopy (SPEM) and Second Harmonic Generation Microscopy (SHGM)

NASA Astrophysics Data System (ADS)

Putzeys, T.; Wübbenhorst, M.; van der Veen, M. A.

2015-06-01

Bio-organic materials such as bones, teeth, and tendon generally show nonlinear optical (Masters and So in Handbook of Biomedical Nonlinear Optical Microscopy, 2008), pyro- and piezoelectric (Fukada and Yasuda in J Phys Soc Jpn 12:1158, 1957) properties, implying a permanent polarization, the presence of which can be rationalized by describing the growth of the sample and the creation of a polar axis according to Markov's theory of stochastic processes (Hulliger in Biophys J 84:3501, 2003; Batagiannis et al. in Curr Opin Solid State Mater Sci 17:107, 2010). Two proven, versatile techniques for probing spontaneous polarization distributions in solids are scanning pyroelectric microscopy (SPEM) and second harmonic generation microscopy (SHGM). The combination of pyroelectric scanning with SHG-microscopy in a single experimental setup leading to complementary pyroelectric and nonlinear optical data is demonstrated, providing us with a more complete image of the polarization in organic materials. Crystals consisting of a known polar and hyperpolarizable material, CNS (4-chloro-4-nitrostilbene) are used as a reference sample, to verify the functionality of the setup, with both SPEM and SHGM images revealing the same polarization domain information. In contrast, feline and human nails exhibit a pyroelectric response, but a second harmonic response is absent for both keratin containing materials, implying that there may be symmetry-allowed SHG, but with very inefficient second harmonophores. This new approach to polarity detection provides additional information on the polar and hyperpolar nature in a variety of (bio) materials.

MAMMALIAN APOPTOSIS IN WHOLE NEONATAL OVARIES, EMBRYOS AND FETAL LIMBS USING CONFOCAL MICROSCOPY

EPA Science Inventory

The emergence of confocal laser scanning microscopy (CLSM) as a technique capable of optically generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure offers a viable alternative to traditional section...

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

NASA Astrophysics Data System (ADS)

Ryseva, Ekaterina; Zhukova, Ekaterina

2013-05-01

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

Confocal microscopy studies of morphology and apoptosis: ovaries, limbs, embryos and insects

EPA Science Inventory

Confocal laser scanning microscopy (CLSM) is a technique that is capable of generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure. In many ways CLSM offers an alternative to traditional sectioning ap...

Confocal microscopy of thick tissue sections: 3D visualizaiton of rat kidney glomeruli

EPA Science Inventory

Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

Confocal Microscopy of thick tissue sections: 3D Visualization of rat kidney glomeruli

EPA Science Inventory

Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

Novel Electrochemical Process for Treatment of Perchlorate in Waste Water

DTIC Science & Technology

2011-03-06

Prepared in Different Processes: (b) in 0.1 M Pyrrole Solution with 0.1 M NaCl at 0.8 V for 20 min; (c) at 0.5 V for 400 s in 0.1 M ClO4- Solution and...polypyrrole Py pyrrole SEM scanning electron microscopy SON statement of need XPS X-ray photoelectron spectroscopy v Acknowledgments This work is...shows the scanning electron microscopy (SEM) images of carbon fiber paper and a CNT array grown on carbon fiber paper. Pyrrole (Py) deposition

Helix handedness of Leptospira interrogans as determined by scanning electron microscopy.

PubMed Central

Carleton, O; Charon, N W; Allender, P; O'Brien, S

1979-01-01

Representative serovars and strains of the seven genetic groups of Leptospira interrogans, and two previously studied serovars, were all found to form exclusively right-handed helices as determined by scanning electron microscopy. No change in handedness occurred in cells grown in a minimal medium (Tween-80 albumin) compared to cells grown in a rich medium (rabbit serum). The right-handedness of the organisms was related to the evolution, cell wall structure, and the mechanism of motility of L. interrogans. Images PMID:438122

Modular Scanning Confocal Microscope with Digital Image Processing

PubMed Central

McCluskey, Matthew D.

2016-01-01

In conventional confocal microscopy, a physical pinhole is placed at the image plane prior to the detector to limit the observation volume. In this work, we present a modular design of a scanning confocal microscope which uses a CCD camera to replace the physical pinhole for materials science applications. Experimental scans were performed on a microscope resolution target, a semiconductor chip carrier, and a piece of etched silicon wafer. The data collected by the CCD were processed to yield images of the specimen. By selecting effective pixels in the recorded CCD images, a virtual pinhole is created. By analyzing the image moments of the imaging data, a lateral resolution enhancement is achieved by using a 20 × / NA = 0.4 microscope objective at 532 nm laser wavelength. PMID:27829052

PtyNAMi: ptychographic nano-analytical microscope at PETRA III: interferometrically tracking positions for 3D x-ray scanning microscopy using a ball-lens retroreflector

NASA Astrophysics Data System (ADS)

Schroer, Christian G.; Seyrich, Martin; Kahnt, Maik; Botta, Stephan; Döhrmann, Ralph; Falkenberg, Gerald; Garrevoet, Jan; Lyubomirskiy, Mikhail; Scholz, Maria; Schropp, Andreas; Wittwer, Felix

2017-09-01

In recent years, ptychography has revolutionized x-ray microscopy in that it is able to overcome the diffraction limit of x-ray optics, pushing the spatial resolution limit down to a few nanometers. However, due to the weak interaction of x rays with matter, the detection of small features inside a sample requires a high coherent fluence on the sample, a high degree of mechanical stability, and a low background signal from the x-ray microscope. The x-ray scanning microscope PtyNAMi at PETRA III is designed for high-spatial-resolution 3D imaging with high sensitivity. The design concept is presented with a special focus on real-time metrology of the sample position during tomographic scanning microscopy.

3D fluorescence anisotropy imaging using selective plane illumination microscopy.

PubMed

Hedde, Per Niklas; Ranjit, Suman; Gratton, Enrico

2015-08-24

Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein.

Thermal radiation scanning tunnelling microscopy

NASA Astrophysics Data System (ADS)

de Wilde, Yannick; Formanek, Florian; Carminati, Rémi; Gralak, Boris; Lemoine, Paul-Arthur; Joulain, Karl; Mulet, Jean-Philippe; Chen, Yong; Greffet, Jean-Jacques

2006-12-01

In standard near-field scanning optical microscopy (NSOM), a subwavelength probe acts as an optical `stethoscope' to map the near field produced at the sample surface by external illumination. This technique has been applied using visible, infrared, terahertz and gigahertz radiation to illuminate the sample, providing a resolution well beyond the diffraction limit. NSOM is well suited to study surface waves such as surface plasmons or surface-phonon polaritons. Using an aperture NSOM with visible laser illumination, a near-field interference pattern around a corral structure has been observed, whose features were similar to the scanning tunnelling microscope image of the electronic waves in a quantum corral. Here we describe an infrared NSOM that operates without any external illumination: it is a near-field analogue of a night-vision camera, making use of the thermal infrared evanescent fields emitted by the surface, and behaves as an optical scanning tunnelling microscope. We therefore term this instrument a `thermal radiation scanning tunnelling microscope' (TRSTM). We show the first TRSTM images of thermally excited surface plasmons, and demonstrate spatial coherence effects in near-field thermal emission.

A joint Richardson-Lucy deconvolution algorithm for the reconstruction of multifocal structured illumination microscopy data.

PubMed

Ströhl, Florian; Kaminski, Clemens F

2015-01-16

We demonstrate the reconstruction of images obtained by multifocal structured illumination microscopy, MSIM, using a joint Richardson-Lucy, jRL-MSIM, deconvolution algorithm, which is based on an underlying widefield image-formation model. The method is efficient in the suppression of out-of-focus light and greatly improves image contrast and resolution. Furthermore, it is particularly well suited for the processing of noise corrupted data. The principle is verified on simulated as well as experimental data and a comparison of the jRL-MSIM approach with the standard reconstruction procedure, which is based on image scanning microscopy, ISM, is made. Our algorithm is efficient and freely available in a user friendly software package.

A joint Richardson—Lucy deconvolution algorithm for the reconstruction of multifocal structured illumination microscopy data

NASA Astrophysics Data System (ADS)

Ströhl, Florian; Kaminski, Clemens F.

2015-03-01

We demonstrate the reconstruction of images obtained by multifocal structured illumination microscopy, MSIM, using a joint Richardson-Lucy, jRL-MSIM, deconvolution algorithm, which is based on an underlying widefield image-formation model. The method is efficient in the suppression of out-of-focus light and greatly improves image contrast and resolution. Furthermore, it is particularly well suited for the processing of noise corrupted data. The principle is verified on simulated as well as experimental data and a comparison of the jRL-MSIM approach with the standard reconstruction procedure, which is based on image scanning microscopy, ISM, is made. Our algorithm is efficient and freely available in a user friendly software package.

Evaluation of environmental scanning electron microscopy for analysis of Proteus mirabilis crystalline biofilms in situ on urinary catheters

PubMed Central

Holling, Nina; Dedi, Cinzia; Jones, Caroline E; Hawthorne, Joseph A; Hanlon, Geoffrey W; Salvage, Jonathan P; Patel, Bhavik A; Barnes, Lara M; Jones, Brian V

2014-01-01

Proteus mirabilis is a common cause of catheter-associated urinary tract infections and frequently leads to blockage of catheters due to crystalline biofilm formation. Scanning electron microscopy (SEM) has proven to be a valuable tool in the study of these unusual biofilms, but entails laborious sample preparation that can introduce artefacts, undermining the investigation of biofilm development. In contrast, environmental scanning electron microscopy (ESEM) permits imaging of unprocessed, fully hydrated samples, which may provide much insight into the development of P. mirabilis biofilms. Here, we evaluate the utility of ESEM for the study of P. mirabilis crystalline biofilms in situ, on urinary catheters. In doing so, we compare this to commonly used conventional SEM approaches for sample preparation and imaging. Overall, ESEM provided excellent resolution of biofilms formed on urinary catheters and revealed structures not observed in standard SEM imaging or previously described in other studies of these biofilms. In addition, we show that energy-dispersive X-ray spectroscopy (EDS) may be employed in conjunction with ESEM to provide information regarding the elemental composition of crystalline structures and demonstrate the potential for ESEM in combination with EDS to constitute a useful tool in exploring the mechanisms underpinning crystalline biofilm formation. PMID:24786314

Electron Microscopy Localization and Characterization of Functionalized Composite Organic-Inorganic SERS Nanoparticles on Leukemia Cells

PubMed Central

Koh, Ai Leen; Shachaf, Catherine M.; Elchuri, Sailaja; Nolan, Garry P.; Sinclair, Robert

2008-01-01

We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet Scanning Electron Microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron detector (BSE) was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution Transmission Electron Microscope (TEM) images and Scanning Auger Electron Spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens. PMID:18995965

Advances in imaging and quantification of electrical properties at the nanoscale using Scanning Microwave Impedance Microscopy (sMIM)

NASA Astrophysics Data System (ADS)

Friedman, Stuart; Stanke, Fred; Yang, Yongliang; Amster, Oskar

Scanning Microwave Impedance Microscopy (sMIM) is a mode for Atomic Force Microscopy (AFM) enabling imaging of unique contrast mechanisms and measurement of local permittivity and conductivity at the 10's of nm length scale. sMIM has been applied to a variety of systems including nanotubes, nanowires, 2D materials, photovoltaics and semiconductor devices. Early results were largely semi-quantitative. This talk will focus on techniques for extracting quantitative physical parameters such as permittivity, conductivity, doping concentrations and thin film properties from sMIM data. Particular attention will be paid to non-linear materials where sMIM has been used to acquire nano-scale capacitance-voltage curves. These curves can be used to identify the dopant type (n vs p) and doping level in doped semiconductors, both bulk samples and devices. Supported in part by DOE-SBIR DE-SC0009856.

High-angle annular dark field scanning transmission electron microscopy on carbon-based functional polymer systems.

PubMed

Sourty, Erwan; van Bavel, Svetlana; Lu, Kangbo; Guerra, Ralph; Bar, Georg; Loos, Joachim

2009-06-01

Two purely carbon-based functional polymer systems were investigated by bright-field conventional transmission electron microscopy (CTEM) and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). For a carbon black (CB) filled polymer system, HAADF-STEM provides high contrast between the CB agglomerates and the polymer matrix so that details of the interface organization easily can be revealed and assignment of the CB phase is straightforward. For a second system, the functional polymer blend representing the photoactive layer of a polymer solar cell, details of its nanoscale organization could be observed that were not accessible with CTEM. By varying the camera length in HAADF-STEM imaging, the contrast can be enhanced between crystalline and amorphous compounds due to diffraction contrast so that nanoscale interconnections between domains are identified. In general, due to its incoherent imaging characteristics HAADF-STEM allows for reliable interpretation of the data obtained.

Two-dimensional dopant profiling of gallium nitride p-n junctions by scanning capacitance microscopy

NASA Astrophysics Data System (ADS)

Lamhamdi, M.; Cayrel, F.; Frayssinet, E.; Bazin, A. E.; Yvon, A.; Collard, E.; Cordier, Y.; Alquier, D.

2016-04-01

Two-dimensional imaging of dopant profiles for n and p-type regions are relevant for the development of new power semiconductors, especially for gallium nitride (GaN) for which classical profiling techniques are not adapted. This is a challenging task since it needs a technique with simultaneously good sensitivity, high spatial resolution and high dopant gradient resolution. To face these challenges, scanning capacitance microscopy combined with Atomic Force Microscopy is a good candidate, presenting reproducible results, as demonstrated in literature. In this work, we attempt to distinguish reliably and qualitatively the various doping concentrations and type at p-n and unipolar junctions. For both p-n and unipolar junctions three kinds of samples were prepared and measured separately. The space-charge region of the p-n metallurgical junction, giving rise to different contrasts under SCM imaging, is clearly observed, enlightening the interest of the SCM technique.

Imaging plasmodesmata with high-resolution scanning electron microscopy.

PubMed

Barton, Deborah A; Overall, Robyn L

2015-01-01

High-resolution scanning electron microscopy (HRSEM) is an effective tool to investigate the distribution of plasmodesmata within plant cell walls as well as to probe their complex, three-dimensional architecture. It is a useful alternative to traditional transmission electron microscopy (TEM) in which plasmodesmata are sectioned to reveal their internal substructures. Benefits of adopting an HRSEM approach to studies of plasmodesmata are that the specimen preparation methods are less complex and time consuming than for TEM, many plasmodesmata within a large region of tissue can be imaged in a single session, and three-dimensional information is readily available without the need for reconstructing TEM serial sections or employing transmission electron tomography, both of which are lengthy processes. Here we describe methods to prepare plant samples for HRSEM using pre- or postfixation extraction of cellular material in order to visualize plasmodesmata embedded within plant cell walls.

Three-dimensional Imaging and Scanning: Current and Future Applications for Pathology

PubMed Central

Farahani, Navid; Braun, Alex; Jutt, Dylan; Huffman, Todd; Reder, Nick; Liu, Zheng; Yagi, Yukako; Pantanowitz, Liron

2017-01-01

Imaging is vital for the assessment of physiologic and phenotypic details. In the past, biomedical imaging was heavily reliant on analog, low-throughput methods, which would produce two-dimensional images. However, newer, digital, and high-throughput three-dimensional (3D) imaging methods, which rely on computer vision and computer graphics, are transforming the way biomedical professionals practice. 3D imaging has been useful in diagnostic, prognostic, and therapeutic decision-making for the medical and biomedical professions. Herein, we summarize current imaging methods that enable optimal 3D histopathologic reconstruction: Scanning, 3D scanning, and whole slide imaging. Briefly mentioned are emerging platforms, which combine robotics, sectioning, and imaging in their pursuit to digitize and automate the entire microscopy workflow. Finally, both current and emerging 3D imaging methods are discussed in relation to current and future applications within the context of pathology. PMID:28966836

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.

Characterisation of a resolution enhancing image inversion interferometer.

PubMed

Wicker, Kai; Sindbert, Simon; Heintzmann, Rainer

2009-08-31

Image inversion interferometers have the potential to significantly enhance the lateral resolution and light efficiency of scanning fluorescence microscopes. Self-interference of a point source's coherent point spread function with its inverted copy leads to a reduction in the integrated signal for off-axis sources compared to sources on the inversion axis. This can be used to enhance the resolution in a confocal laser scanning microscope. We present a simple image inversion interferometer relying solely on reflections off planar surfaces. Measurements of the detection point spread function for several types of light sources confirm the predicted performance and suggest its usability for scanning confocal fluorescence microscopy.

Scanning Electron Microscopy (SEM) Procedure for HE Powders on a Zeiss Sigma HD VP SEM

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

Zaka, F.

This method describes the characterization of inert and HE materials by the Zeiss Sigma HD VP field emission Scanning Electron Microscope (SEM). The SEM uses an accelerated electron beam to generate high-magnification images of explosives and other materials. It is fitted with five detectors (SE, Inlens, STEM, VPSE, HDBSD) to enable imaging of the sample via different secondary electron signatures, angles, and energies. In addition to imaging through electron detection, the microscope is also fitted with two Oxford Instrument Energy Dispersive Spectrometer (EDS) 80 mm detectors to generate elemental constituent spectra and two-dimensional maps of the material being scanned.

The influence of C s/C c correction in analytical imaging and spectroscopy in scanning and transmission electron microscopy

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

Zaluzec, Nestor J.

Aberration correction in scanning/transmission electron microscopy (S/TEM) owes much to the efforts of a small dedicated group of innovators. Leading that frontier has been Prof. Harald Rose. To date his leadership and dynamic personality has spearheaded our ability to leave behind many of the limitations imposed by spherical aberration (C s) in high resolution phase contrast imaging. Following shortly behind, has been the development of chromatic aberration correction (C c) which augments those accomplishments. In this study we will review and summarize how the combination of C s/C c technology enhances our ability to conduct hyperspectral imaging and spectroscopy inmore » today's and future computationally mediated experiments in both thin as well as realistic specimens in vacuo and during in-situ/environmental experiments.« less

The influence of C s/C c correction in analytical imaging and spectroscopy in scanning and transmission electron microscopy

DOE PAGES

Zaluzec, Nestor J.

2014-11-11

Aberration correction in scanning/transmission electron microscopy (S/TEM) owes much to the efforts of a small dedicated group of innovators. Leading that frontier has been Prof. Harald Rose. To date his leadership and dynamic personality has spearheaded our ability to leave behind many of the limitations imposed by spherical aberration (C s) in high resolution phase contrast imaging. Following shortly behind, has been the development of chromatic aberration correction (C c) which augments those accomplishments. In this study we will review and summarize how the combination of C s/C c technology enhances our ability to conduct hyperspectral imaging and spectroscopy inmore » today's and future computationally mediated experiments in both thin as well as realistic specimens in vacuo and during in-situ/environmental experiments.« less

Scanning tunneling microscopy and atomic force microscopy: application to biology and technology.

PubMed

Hansma, P K; Elings, V B; Marti, O; Bracker, C E

1988-10-14

The scanning tunneling microscope (STM) and the atomic force microscope (AFM) are scanning probe microscopes capable of resolving surface detail down to the atomic level. The potential of these microscopes for revealing subtle details of structure is illustrated by atomic resolution images including graphite, an organic conductor, an insulating layered compound, and individual adsorbed oxygen atoms on a semiconductor. Application of the STM for imaging biological materials directly has been hampered by the poor electron conductivity of most biological samples. The use of thin conductive metal coatings and replicas has made it possible to image some biological samples, as indicated by recently obtained images of a recA-DNA complex, a phospholipid bilayer, and an enzyme crystal. The potential of the AFM, which does not require a conductive sample, is shown with molecular resolution images of a nonconducting organic monolayer and an amino acid crystal that reveals individual methyl groups on the ends of the amino acids. Applications of these new microscopes to technology are demonstrated with images of an optical disk stamper, a diffraction grating, a thin-film magnetic recording head, and a diamond cutting tool. The STM has even been used to improve the quality of diffraction gratings and magnetic recording heads.

STEMsalabim: A high-performance computing cluster friendly code for scanning transmission electron microscopy image simulations of thin specimens.

PubMed

Oelerich, Jan Oliver; Duschek, Lennart; Belz, Jürgen; Beyer, Andreas; Baranovskii, Sergei D; Volz, Kerstin

2017-06-01

We present a new multislice code for the computer simulation of scanning transmission electron microscope (STEM) images based on the frozen lattice approximation. Unlike existing software packages, the code is optimized to perform well on highly parallelized computing clusters, combining distributed and shared memory architectures. This enables efficient calculation of large lateral scanning areas of the specimen within the frozen lattice approximation and fine-grained sweeps of parameter space. Copyright © 2017 Elsevier B.V. All rights reserved.

Big Data and Deep data in scanning and electron microscopies: functionality from multidimensional data sets

DOE PAGES

Belianinov, Alex; Vasudevan, Rama K; Strelcov, Evgheni; ...

2015-05-13

The development of electron, and scanning probe microscopies in the second half of the twentieth century have produced spectacular images of internal structure and composition of matter with, at nanometer, molecular, and atomic resolution. Largely, this progress was enabled by computer-assisted methods of microscope operation, data acquisition and analysis. The progress in imaging technologies in the beginning of the twenty first century has opened the proverbial floodgates of high-veracity information on structure and functionality. High resolution imaging now allows information on atomic positions with picometer precision, allowing for quantitative measurements of individual bond length and angles. Functional imaging often leadsmore » to multidimensional data sets containing partial or full information on properties of interest, acquired as a function of multiple parameters (time, temperature, or other external stimuli). Here, we review several recent applications of the big and deep data analysis methods to visualize, compress, and translate this data into physically and chemically relevant information from imaging data.« less

Big Data and Deep data in scanning and electron microscopies: functionality from multidimensional data sets

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

Belianinov, Alex; Vasudevan, Rama K; Strelcov, Evgheni

The development of electron, and scanning probe microscopies in the second half of the twentieth century have produced spectacular images of internal structure and composition of matter with, at nanometer, molecular, and atomic resolution. Largely, this progress was enabled by computer-assisted methods of microscope operation, data acquisition and analysis. The progress in imaging technologies in the beginning of the twenty first century has opened the proverbial floodgates of high-veracity information on structure and functionality. High resolution imaging now allows information on atomic positions with picometer precision, allowing for quantitative measurements of individual bond length and angles. Functional imaging often leadsmore » to multidimensional data sets containing partial or full information on properties of interest, acquired as a function of multiple parameters (time, temperature, or other external stimuli). Here, we review several recent applications of the big and deep data analysis methods to visualize, compress, and translate this data into physically and chemically relevant information from imaging data.« less

Development of a surface topography instrument for automotive textured steel plate

NASA Astrophysics Data System (ADS)

Wang, Zhen; Wang, Shenghuai; Chen, Yurong; Xie, Tiebang

2010-08-01

The surface topography of automotive steel plate is decisive to its stamping, painting and image clarity performances. For measuring this kind of surface topography, an instrument has been developed based on the principle of vertical scanning white light microscopy interference principle. The microscopy interference system of this instrument is designed based on the structure of Linnik interference microscopy. The 1D worktable of Z direction is designed and introduced in details. The work principle of this instrument is analyzed. In measuring process, the interference microscopy is derived as a whole and the measured surface is scanned in vertical direction. The measurement accuracy and validity is verified by templates. Surface topography of textured steel plate is also measured by this instrument.

Image Analysis, Microscopic, and Spectrochemical Study of the PVC Dry Blending Process,

DTIC Science & Technology

The dry blending process used in the production of electrical grade pvc formulations has been studies using a combination of image analysis , microscopic...by image analysis techniques. Optical and scanning electron microscopy were used to assess morphological differences. Spectrochemical techniques were used to indicate chemical changes.

Fast axial scanning for 2-photon microscopy using liquid lens technology.

PubMed

Tehrani, Kayvan Forouhesh; Sun, Min Kyoung; Karumbaiah, Lohitash; Mortensen, Luke J

2017-03-01

Scanning microscopy methods require movement of the focus in Z coordinates to produce an image of a 3-dimensional volume. In a typical imaging system, the optical setup is kept fixed and either the sample or the objective is translated with a mechanical stage driven by a stepper motor or a piezoelectric element. Mechanical Z scanning is precise, but its slow response and vulnerability to mechanical vibrations and stress make it disadvantageous to image dynamic, time-varying samples such as live cell structures. An alternative method less susceptible to these problems is to change the focal plane using conjugate optics. Deformable mirrors, acoustooptics, and electrically tunable lenses have been experimented with to achieve this goal and have attained very fast and precise Z-scanning without physically moving the sample. Here, we present the use of a liquid lens for fast axial scanning. Liquid lenses have a long functional life, high degree of phase shift, and low sensitivity to mechanical stress. They work on the principle of refraction at a liquid-liquid interface. At the boundary of a polar and an apolar liquid a spherical surface is formed whose curvature can be controlled by adjusting its relative wettability using electrowetting. We characterize the effects of the lens on attainable Z displacement, beam spectral characteristics, and pulse duration as compared with mechanical scanning.

Fast axial scanning for 2-photon microscopy using liquid lens technology

PubMed Central

Tehrani, Kayvan Forouhesh; Sun, Min Kyoung; Karumbaiah, Lohitash; Mortensen, Luke J.

2018-01-01

Scanning microscopy methods require movement of the focus in Z coordinates to produce an image of a 3-dimensional volume. In a typical imaging system, the optical setup is kept fixed and either the sample or the objective is translated with a mechanical stage driven by a stepper motor or a piezoelectric element. Mechanical Z scanning is precise, but its slow response and vulnerability to mechanical vibrations and stress make it disadvantageous to image dynamic, time-varying samples such as live cell structures. An alternative method less susceptible to these problems is to change the focal plane using conjugate optics. Deformable mirrors, acoustooptics, and electrically tunable lenses have been experimented with to achieve this goal and have attained very fast and precise Z-scanning without physically moving the sample. Here, we present the use of a liquid lens for fast axial scanning. Liquid lenses have a long functional life, high degree of phase shift, and low sensitivity to mechanical stress. They work on the principle of refraction at a liquid-liquid interface. At the boundary of a polar and an apolar liquid a spherical surface is formed whose curvature can be controlled by adjusting its relative wettability using electrowetting. We characterize the effects of the lens on attainable Z displacement, beam spectral characteristics, and pulse duration as compared with mechanical scanning. PMID:29706682

Comparison of divided and full pupil configurations for line-scanning confocal microscopy in human skin and oral mucosa

NASA Astrophysics Data System (ADS)

Larson, Bjorg; Abeytunge, Sanjeewa; Glazowski, Chris; Rajadhyaksha, Milind

2012-02-01

Confocal point-scanning microscopy has been showing promise in the detection, diagnosing and mapping of skin lesions in clinical settings. The noninvasive technique allows provides optical sectioning and cellular resolution for in vivo diagnosis of melanoma and basal cell carcinoma and pre-operative and intra-operative mapping of margins. The imaging has also enabled more accurate "guided" biopsies while minimizing the otherwise large number of "blind" biopsies. Despite these translational advances, however, point-scanning technology remains relatively complex and expensive. Line-scanning technology may offer an alternative approach to accelerate translation to the clinic. Line-scanning, using fewer optical components, inexpensive linear-array detectors and custom electronics, may enable smaller, simpler and lower-cost confocal microscopes. A line is formed using a cylindrical lens and scanned through the back focal plane of the objective with a galvanometric scanner. A linear CCD is used for detection. Two pupil configurations were compared for performance in imaging human tissue. In the full-pupil configuration, illumination and detection is made through the full objective pupil. In the divided pupil approach, half the pupil is illuminated and the other half is used for detection. The divided pupil configuration loses spatial and axial resolution due to a diminished NA, but the sectioning capability and rejection of background is improved. Imaging in skin and oral mucosa illustrate the performance of the two configurations.

Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution.

PubMed

Lillis, Kyle P; Eng, Alfred; White, John A; Mertz, Jerome

2008-07-30

We describe a simple two-photon fluorescence imaging strategy, called targeted path scanning (TPS), to monitor the dynamics of spatially extended neuronal networks with high spatiotemporal resolution. Our strategy combines the advantages of mirror-based scanning, minimized dead time, ease of implementation, and compatibility with high-resolution low-magnification objectives. To demonstrate the performance of TPS, we monitor the calcium dynamics distributed across an entire juvenile rat hippocampus (>1.5mm), at scan rates of 100 Hz, with single cell resolution and single action potential sensitivity. Our strategy for fast, efficient two-photon microscopy over spatially extended regions provides a particularly attractive solution for monitoring neuronal population activity in thick tissue, without sacrificing the signal-to-noise ratio or high spatial resolution associated with standard two-photon microscopy. Finally, we provide the code to make our technique generally available.

Ultra-thin resin embedding method for scanning electron microscopy of individual cells on high and low aspect ratio 3D nanostructures.

PubMed

Belu, A; Schnitker, J; Bertazzo, S; Neumann, E; Mayer, D; Offenhäusser, A; Santoro, F

2016-07-01

The preparation of biological cells for either scanning or transmission electron microscopy requires a complex process of fixation, dehydration and drying. Critical point drying is commonly used for samples investigated with a scanning electron beam, whereas resin-infiltration is typically used for transmission electron microscopy. Critical point drying may cause cracks at the cellular surface and a sponge-like morphology of nondistinguishable intracellular compartments. Resin-infiltrated biological samples result in a solid block of resin, which can be further processed by mechanical sectioning, however that does not allow a top view examination of small cell-cell and cell-surface contacts. Here, we propose a method for removing resin excess on biological samples before effective polymerization. In this way the cells result to be embedded in an ultra-thin layer of epoxy resin. This novel method highlights in contrast to standard methods the imaging of individual cells not only on nanostructured planar surfaces but also on topologically challenging substrates with high aspect ratio three-dimensional features by scanning electron microscopy. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

New methods for image collection and analysis in scanning Auger microscopy

NASA Technical Reports Server (NTRS)

Browning, R.

1985-01-01

While scanning Auger micrographs are used extensively for illustrating the stoichiometry of complex surfaces and for indicating areas of interest for fine point Auger spectroscopy, there are many problems in the quantification and analysis of Auger images. These problems include multiple contrast mechanisms and the lack of meaningful relationships with other Auger data. Collection of multielemental Auger images allows some new approaches to image analysis and presentation. Information about the distribution and quantity of elemental combinations at a surface are retrievable, and particular combinations of elements can be imaged, such as alloy phases. Results from the precipitate hardened alloy Al-2124 illustrate multispectral Auger imaging.

Multi-imaging analysis of nascent surface structures generated during femtosecond laser irradiation of silicon in high vacuum

NASA Astrophysics Data System (ADS)

Gesuele, F.; JJ Nivas, J.; Fittipaldi, R.; Altucci, C.; Bruzzese, R.; Maddalena, P.; Amoruso, S.

2018-02-01

We report a correlative imaging analysis of a crystalline silicon target after irradiation with a low number of 1055 nm, 850 fs laser pulses with several microscopy techniques (e.g., scanning electron microscopy, atomic force microscopy, Raman micro-imaging and confocal optical microscopy). The analysis is carried out on samples irradiated both in high vacuum and at atmospheric pressure conditions, evidencing interesting differences induced by the ambient environment. In high-vacuum conditions, the results evidence the formation of a halo, which is constituted by alternate stripes of amorphous and crystalline silicon, around the nascent ablation crater. In air, such an effect is drastically reduced, due to the significant back-deposition of nanoparticulate material induced by the larger ambient pressure.

Study of electromechanical and mechanical properties of bacteria using force microscopy

NASA Astrophysics Data System (ADS)

Reukov, Vladimir; Thompson, Gary; Nikiforov, Maxim; Guo, Senli; Ovchinnikov, Oleg; Jesse, Stephen; Kalinin, Sergei; Vertegel, Alexey

2010-03-01

The application of scanning probe microscopy (SPM) to biological systems has evolved over the past decade into a multimodal and spectroscopic instrument that provides multiple information channels at each spatial pixel acquired. Recently, functional recognition imaging based on differing electromechanical properties between Gram negative and Gram positive bacteria was achieved using artificial neural network analysis of band excitation piezoresponse force microscopy (BEPFM) data. The immediate goal of this project was to study mechanical and electromechanical properties of bacterial systems physiologically-relevant solutions using Band-width Excitation Piezoresponce Force Microscopy (BE PFM) in combination with Force Mapping. Electromechanical imaging in physiological environments will improve the versatility of functional recognition imaging and open the way for application of the rapid BEPFM line mode method to other living cell systems.

Field Emission Auger Electron Spectroscopy with Scanning Auger Microscopy |

Science.gov Websites

0.5 at.% for elements from lithium to uranium. Depth Profiling Removes successive layers by using size (> ~25 nm). Imaging Obtains SEM micrographs with up to 20,000x magnification by using raster scanning with a highly focused electron beam ≥25 nm in diameter. Using the same raster scan, SAM can

New Insights on Subsurface Imaging of Carbon Nanotubes in Polymer Composites via Scanning Electron Microscopy

NASA Technical Reports Server (NTRS)

Zhao, Minhua; Ming, Bin; Kim, Jae-Woo; Gibbons, Luke J.; Gu, Xiaohong; Nguyen, Tinh; Park, Cheol; Lillehei, Peter T.; Villarrubia, J. S.; Vladar, Andras E.;

Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscope.

PubMed

Peckys, Diana B; Veith, Gabriel M; Joy, David C; de Jonge, Niels

2009-12-14

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.

Surface-polariton propagation for scanning near-field optical microscopy application.

PubMed

Keilmann, F

1999-01-01

Surface plasmon-, phonon- and exciton-polaritons exist on specific materials in specific spectral regions. We assess the properties of such travelling surface-bound electromagnetic waves relevant for scanning near-field optical microscopy applications, i.e. the tightness of surface binding, the attenuation, the phase velocity and the coupling with free-space electromagnetic waves. These quantities can be directly determined by photographic imaging of surface plasmon- and surface phonon-polaritons, in both the visible and mid-infared regions. Focusing of mid-infrared surface plasmons is demonstrated. Surface waveguides to transport and focus photons to the tip of a scanning near-field probe are outlined.

Scanning transmission X-ray, laser scanning, and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms.

PubMed

Lawrence, J R; Swerhone, G D W; Leppard, G G; Araki, T; Zhang, X; West, M M; Hitchcock, A P

2003-09-01

Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.

Sample preparation methods for scanning electron microscopy of homogenized Al-Mg-Si billets: A comparative study

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

Österreicher, Johannes Albert; Kumar, Manoj

Characterization of Mg-Si precipitates is crucial for optimizing the homogenization heat treatment of Al-Mg-Si alloys. Although sample preparation is key for high quality scanning electron microscopy imaging, most common methods lead to dealloying of Mg-Si precipitates. In this article we systematically evaluate different sample preparation methods: mechanical polishing, etching with various reagents, and electropolishing using different electrolytes. We demonstrate that the use of a nitric acid and methanol electrolyte for electropolishing a homogenized Al-Mg-Si alloy prevents the dissolution of Mg-Si precipitates, resulting in micrographs of higher quality. This preparation method is investigated in depth and the obtained scanning electron microscopymore » images are compared with transmission electron micrographs: the shape and size of Mg-Si precipitates appear very similar in either method. The scanning electron micrographs allow proper identification and measurement of the Mg-Si phases including needles with lengths of roughly 200 nm. These needles are β″ precipitates as confirmed by high resolution transmission electron microscopy. - Highlights: •Secondary precipitation in homogenized 6xxx Al alloys is crucial for extrudability. •Existing sample preparation methods for SEM are improvable. •Electropolishing with nitric acid/methanol yields superior quality in SEM. •The obtained micrographs are compared to TEM micrographs.« less

Hard x-ray scanning imaging achieved with bonded multilayer Laue lenses

DOE PAGES

Huang, Xiaojing; Xu, Weihe; Nazaretski, Evgeny; ...

2017-04-05

Here, we report scanning hard x-ray imaging with a monolithic focusing optic consisting of two multilayer Laue lenses (MLLs) bonded together. With optics pre-characterization and accurate control of the bonding process, we show that a common focal plane for both MLLs can be realized at 9.317 keV. Using bonded MLLs, we obtained a scanning transmission image of a star test pattern with a resolution of 50 × 50 nm 2. By applying a ptychography algorithm, we obtained a probe size of 17 × 38 nm 2 and an object image with a resolution of 13 × 13 nm 2. Finally,more » the significant reduction in alignment complexity for bonded MLLs will greatly extend the application range in both scanning and full-field x-ray microscopies.« less

Hard x-ray scanning imaging achieved with bonded multilayer Laue lenses

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

Huang, Xiaojing; Xu, Weihe; Nazaretski, Evgeny

Here, we report scanning hard x-ray imaging with a monolithic focusing optic consisting of two multilayer Laue lenses (MLLs) bonded together. With optics pre-characterization and accurate control of the bonding process, we show that a common focal plane for both MLLs can be realized at 9.317 keV. Using bonded MLLs, we obtained a scanning transmission image of a star test pattern with a resolution of 50 × 50 nm 2. By applying a ptychography algorithm, we obtained a probe size of 17 × 38 nm 2 and an object image with a resolution of 13 × 13 nm 2. Finally,more » the significant reduction in alignment complexity for bonded MLLs will greatly extend the application range in both scanning and full-field x-ray microscopies.« less

High-Precision Pinpointing of Luminescent Targets in Encoder-Assisted Scanning Microscopy Allowing High-Speed Quantitative Analysis.

PubMed

Zheng, Xianlin; Lu, Yiqing; Zhao, Jiangbo; Zhang, Yuhai; Ren, Wei; Liu, Deming; Lu, Jie; Piper, James A; Leif, Robert C; Liu, Xiaogang; Jin, Dayong

2016-01-19

Compared with routine microscopy imaging of a few analytes at a time, rapid scanning through the whole sample area of a microscope slide to locate every single target object offers many advantages in terms of simplicity, speed, throughput, and potential for robust quantitative analysis. Existing techniques that accommodate solid-phase samples incorporating individual micrometer-sized targets generally rely on digital microscopy and image analysis, with intrinsically low throughput and reliability. Here, we report an advanced on-the-fly stage scanning method to achieve high-precision target location across the whole slide. By integrating X- and Y-axis linear encoders to a motorized stage as the virtual "grids" that provide real-time positional references, we demonstrate an orthogonal scanning automated microscopy (OSAM) technique which can search a coverslip area of 50 × 24 mm(2) in just 5.3 min and locate individual 15 μm lanthanide luminescent microspheres with standard deviations of 1.38 and 1.75 μm in X and Y directions. Alongside implementation of an autofocus unit that compensates the tilt of a slide in the Z-axis in real time, we increase the luminescence detection efficiency by 35% with an improved coefficient of variation. We demonstrate the capability of advanced OSAM for robust quantification of luminescence intensities and lifetimes for a variety of micrometer-scale luminescent targets, specifically single down-shifting and upconversion microspheres, crystalline microplates, and color-barcoded microrods, as well as quantitative suspension array assays of biotinylated-DNA functionalized upconversion nanoparticles.

Rare-earth-doped nanophosphors for multicolor cathodoluminescence nanobioimaging using scanning transmission electron microscopy.

PubMed

Furukawa, Taichi; Fukushima, Shoichiro; Niioka, Hirohiko; Yamamoto, Naoki; Miyake, Jun; Araki, Tsutomu; Hashimoto, Mamoru

2015-05-01

We describe rare-earth-doped nanophosphors (RE-NPs) for biological imaging using cathodoluminescence(CL) microscopy based on scanning transmission electron microscopy (STEM). We report the first demonstration of multicolor CL nanobioimaging using STEM with nanophosphors. The CL spectra of the synthesized nanophosphors (Y2O3∶Eu, Y2O3∶Tb) were sufficiently narrow to be distinguished. From CL images of RE-NPs on an elastic carbon-coated copper grid, the spatial resolution was beyond the diffraction limit of light.Y2O3∶Tb and Y2O3∶Eu RE-NPs showed a remarkable resistance against electron beam exposure even at high acceleration voltage (80 kV) and retained a CL intensity of more than 97% compared with the initial intensity for 1 min. In biological CL imaging with STEM, heavy-metal-stained cell sections containing the RE-NPs were prepared,and both the CL images of RE-NPs and cellular structures, such as mitochondria, were clearly observed from STEM images with high contrast. The cellular CL imaging using RE-NPs also had high spatial resolution even though heavy-metal-stained cells are normally regarded as highly scattering media. Moreover, since theRE-NPs exhibit photoluminescence (PL) excited by UV light, they are useful for multimodal correlative imaging using CL and PL.

Wide-field imaging through scattering media by scattered light fluorescence microscopy

NASA Astrophysics Data System (ADS)

Zhou, Yulan; Li, Xun

2017-08-01

To obtain images through scattering media, scattered light fluorescence (SLF) microscopy that utilizes the optical memory effect has been developed. However, the small field of view (FOV) of SLF microscopy limits its application. In this paper, we have introduced a re-modulation method to achieve wide-field imaging through scattering media by SLF microscopy. In the re-modulation method, to raster scan the focus across the object plane, the incident wavefront is re-modulated via a spatial light modulator (SLM) in the updated phase compensation calculated using the optimized iterative algorithm. Compared with the conventional optical memory effect method, the re-modulation method can greatly increase the FOV of a SLF microscope. With the phase compensation theoretically calculated, the process of updating the phase compensation of a high speed SLM is fast. The re-modulation method does not increase the imaging time. The re-modulation method is, therefore, expected to make SLF microscopy have much wider applications in biology, medicine and physiology.

Towards native-state imaging in biological context in the electron microscope

PubMed Central

Weston, Anne E.; Armer, Hannah E. J.

2009-01-01

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context. PMID:19916039

Super-resolved linear fluorescence localization microscopy using photostable fluorophores: A virtual microscopy study

NASA Astrophysics Data System (ADS)

Birk, Udo; Szczurek, Aleksander; Cremer, Christoph

2017-12-01

Current approaches to overcome the conventional limit of the resolution potential of light microscopy (of about 200 nm for visible light), often suffer from non-linear effects, which render the quantification of the image intensities in the reconstructions difficult, and also affect the quantification of the biological structure under investigation. As an attempt to face these difficulties, we discuss a particular method of localization microscopy which is based on photostable fluorescent dyes. The proposed method can potentially be implemented as a fast alternative for quantitative localization microscopy, circumventing the need for the acquisition of thousands of image frames and complex, highly dye-specific imaging buffers. Although the need for calibration remains in order to extract quantitative data (such as the number of emitters), multispectral approaches are largely facilitated due to the much less stringent requirements on imaging buffers. Furthermore, multispectral acquisitions can be readily obtained using commercial instrumentation such as e.g. the conventional confocal laser scanning microscope.

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy

PubMed Central

Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin

2016-01-01

Objectives We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. Methods We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. Results An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. Conclusions The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis. PMID:27525165

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy.

PubMed

Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin; Sohn, Dae Kyung

2016-07-01

We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis.

Near-field microscopy and fluorescence spectroscopy: application to chromosomes labelled with different fluorophores.

PubMed

Mahieu-Williame, L; Falgayrettes, P; Nativel, L; Gall-Borrut, P; Costa, L; Salehzada, T; Bisbal, C

2010-04-01

We have coupled a spectrophotometer with a scanning near-field optical microscope to obtain, with a single scan, simultaneously scanning near-field optical microscope fluorescence images at different wavelengths as well as topography and transmission images. Extraction of the fluorescence spectra enabled us to decompose the different wavelengths of the fluorescence signals which normally overlap. We thus obtained images of the different fluorescence emissions of acridine orange bound to single or double stranded nucleic acids in human metaphase chromosomes before and after DNAse I or RNAse A treatment. The analysis of these images allowed us to visualize some specific chromatin areas where RNA is associated with DNA showing that such a technique could be used to identify multiple components within a cell.

Super-resolved terahertz microscopy by knife-edge scan

NASA Astrophysics Data System (ADS)

Giliberti, V.; Flammini, M.; Ciano, C.; Pontecorvo, E.; Del Re, E.; Ortolani, M.

2017-08-01

We present a compact, all solid-state THz confocal microscope operating at 0.30 THz that achieves super-resolution by using the knife-edge scan approach. In the final reconstructed image, a lateral resolution of 60 μm ≍ λ/17 is demonstrated when the knife-edge is deep in the near-field of the sample surface. When the knife-edge is lifted up to λ/4 from the sample surface, a certain degree of super-resolution is maintained with a resolution of 0.4 mm, i.e. more than a factor 2 if compared to the diffraction-limited scheme. The present results open an interesting path towards super-resolved imaging with in-depth information that would be peculiar to THz microscopy systems.

Determination of the coalescence temperature of latexes by environmental scanning electron microscopy.

PubMed

Gonzalez, Edurne; Tollan, Christopher; Chuvilin, Andrey; Barandiaran, Maria J; Paulis, Maria

2012-08-01

A new methodology for quantitative characterization of the coalescence process of waterborne polymer dispersion (latex) particles by environmental scanning electron microscopy (ESEM) is proposed. The experimental setup has been developed to provide reproducible latex monolayer depositions, optimized contrast of the latex particles, and a reliable readout of the sample temperature. Quantification of the coalescence process under dry conditions has been performed by image processing based on evaluation of the image autocorrelation function. As a proof of concept the coalescence of two latexes with known and differing glass transition temperatures has been measured. It has been shown that a reproducibility of better than 1.5 °C can be obtained for the measurement of the coalescence temperature.

Microscopy and microanalysis 1996

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

Bailey, G.W.; Corbett, J.M.; Dimlich, R.V.W.

1996-12-31

The Proceedings of this Annual Meeting contain paper of members from the three societies. These proceedings emphasizes the common research interests and attempts to eliminate some unwanted overlap. Topics covered are: microscopic analysis of animals with altered gene expression and in-situ gene and antibody localizations, high-resolution elemental mapping of nucleoprofein interactions, plant biology and pathology, quantitative HREM analysis of perfect and defected materials, computational methods for TEM image analysis, high-resolution FESM in materials research, frontiers in polymer microscopy and microanalysis, oxidation and corrosion, micro XRD and XRF, molecular microspectroscopy and spectral imaging, advances in confocal and multidimensional light microscopy, analyticalmore » electron microscopy in biology, correlative microscopy in biological sciences, grain-boundary microengineering, surfaces and interfaces, telepresence microscopy in education and research, MSA educational outreach, quantitative electron probe microanalysis, frontiers of analytical electron microscopy, critical issues in ceramic microstructures, dynamic organization of the cell, pathology, microbiology, high-resolution biological and cryo SEM, and scanning-probe microscopy.« less

Remote focusing in confocal microscopy by means of a modified Alvarez lens.

PubMed

Bawart, M; Jesacher, A; Bernet, S; Ritsch-Marte, M

2018-06-22

Alvarez lenses are actuated lens-pairs which allow one to tune the optical power by mechanical displacement of subelements. Here, we show that a recently realized modified Alvarez lens design which does not require mechanical actuation can be integrated into a confocal microscope. Instead of mechanically moving them, the sublenses are imaged onto each other in a 4f-configuration, where the lateral image shift leading to a change in optical power is created by a galvo-mirror. The avoidance of mechanical lens shifts leads to a large speed gain for axial (and hence also 3D) image scans compared to classical Alvarez lenses. We demonstrate that the suggested operation principle is compatible with confocal microscopy. In order to optimize the system, we have drawn advantage of the flexibility a liquid-crystal spatial light modulator offers for the implementation. For given specifications, dedicated diffractive optical elements or freeform elements can be used in combination with resonant galvo-scanners or acousto-optic beam deflectors, to achieve even faster z-scans than reported here, reaching video rate. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Virtual k -Space Modulation Optical Microscopy

NASA Astrophysics Data System (ADS)

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

2016-07-01

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

Analysis of multi-channel microscopy: Spectral self-interference, multi-detector confocal and 4Pi systems

NASA Astrophysics Data System (ADS)

Davis, Brynmor J.

Fluorescence microscopy is an important and ubiquitous tool in biological imaging due to the high specificity with which fluorescent molecules can be attached to an organism and the subsequent nondestructive in-vivo imaging allowed. Focused-light microscopies allow three-dimensional fluorescence imaging but their resolution is restricted by diffraction. This effect is particularly limiting in the axial dimension as the diffraction-limited focal volume produced by a lens is more extensive along the optical axis than perpendicular to it. Approaches such as confocal microscopy and 4Pi microscopy have been developed to improve the axial resolution. Spectral Self-Interference Fluorescence Microscopy (SSFM) is another high-axial-resolution technique and is the principal subject of this dissertation. Nanometer-precision localization of a single fluorescent layer has been demonstrated using SSFM. This accuracy compares favorably with the axial resolutions given by confocal and 4Pi systems at similar operating parameters (these resolutions are approximately 350nm and 80nm respectively). This theoretical work analyzes the expected performance of the SSFM system when imaging a general object, i.e. an arbitrary fluorophore density function rather than a single layer. An existing model of SSFM is used in simulations to characterize the system's resolution. Several statistically-based reconstruction methods are applied to show that the expected resolution for SSFM is similar to 4Pi microscopy for a general object but does give very high localization accuracy when the object is known to consist of a limited number of layers. SSFM is then analyzed in a linear systems framework and shown to have strong connections, both physically and mathematically, to a multi-channel 4Pi microscope. Fourier-domain analysis confirms that SSFM cannot be expected to outperform this multi-channel 4Pi instrument. Differences between the channels in spatial-scanning, multi-channel microscopies are then exploited to show that such instruments can operate at a sub-Nyquist scanning rate but still produce images largely free of aliasing effects. Multi-channel analysis is also used to show how light typically discarded in confocal and 4Pi systems can be collected and usefully incorporated into the measured image.

Characterization of conductive nanobiomaterials derived from viral assemblies by low-voltage STEM imaging and Raman scattering

NASA Astrophysics Data System (ADS)

Plascencia-Villa, Germán; Carreño-Fuentes, Liliana; Bahena, Daniel; José-Yacamán, Miguel; Palomares, Laura A.; Ramírez, Octavio T.

2014-09-01

New technologies require the development of novel nanomaterials that need to be fully characterized to achieve their potential. High-resolution low-voltage scanning transmission electron microscopy (STEM) has proven to be a very powerful technique in nanotechnology, but its use for the characterization of nanobiomaterials has been limited. Rotavirus VP6 self-assembles into nanotubular assemblies that possess an intrinsic affinity for Au ions. This property was exploited to produce hybrid nanobiomaterials by the in situ functionalization of recombinant VP6 nanotubes with gold nanoparticles. In this work, Raman spectroscopy and advanced analytical electron microscopy imaging with spherical aberration-corrected (Cs) STEM and nanodiffraction at low-voltage doses were employed to characterize nanobiomaterials. STEM imaging revealed the precise structure and arrangement of the protein templates, as well as the nanostructure and atomic arrangement of gold nanoparticles with high spatial sub-Angstrom resolution and avoided radiation damage. The imaging was coupled with backscattered electron imaging, ultra-high resolution scanning electron microscopy and x-ray spectroscopy. The hybrid nanobiomaterials that were obtained showed unique properties as bioelectronic conductive devices and showed enhanced Raman scattering by their precise arrangement into superlattices, displaying the utility of viral assemblies as functional integrative self-assembled nanomaterials for novel applications.

3D visualization and quantification of bone and teeth mineralization for the study of osteo/dentinogenesis in mice models

NASA Astrophysics Data System (ADS)

Marchadier, A.; Vidal, C.; Ordureau, S.; Lédée, R.; Léger, C.; Young, M.; Goldberg, M.

2011-03-01

Research on bone and teeth mineralization in animal models is critical for understanding human pathologies. Genetically modified mice represent highly valuable models for the study of osteo/dentinogenesis defects and osteoporosis. Current investigations on mice dental and skeletal phenotype use destructive and time consuming methods such as histology and scanning microscopy. Micro-CT imaging is quicker and provides high resolution qualitative phenotypic description. However reliable quantification of mineralization processes in mouse bone and teeth are still lacking. We have established novel CT imaging-based software for accurate qualitative and quantitative analysis of mouse mandibular bone and molars. Data were obtained from mandibles of mice lacking the Fibromodulin gene which is involved in mineralization processes. Mandibles were imaged with a micro-CT originally devoted to industrial applications (Viscom, X8060 NDT). 3D advanced visualization was performed using the VoxBox software (UsefulProgress) with ray casting algorithms. Comparison between control and defective mice mandibles was made by applying the same transfer function for each 3D data, thus allowing to detect shape, colour and density discrepencies. The 2D images of transverse slices of mandible and teeth were similar and even more accurate than those obtained with scanning electron microscopy. Image processing of the molars allowed the 3D reconstruction of the pulp chamber, providing a unique tool for the quantitative evaluation of dentinogenesis. This new method is highly powerful for the study of oro-facial mineralizations defects in mice models, complementary and even competitive to current histological and scanning microscopy appoaches.

The surface topography of the choroid plexus. Environmental, low and high vacuum scanning electron microscopy.

PubMed

Mestres, Pedro; Pütz, Norbert; Garcia Gómez de Las Heras, Soledad; García Poblete, Eduardo; Morguet, Andrea; Laue, Michael

2011-05-01

Environmental scanning electron microscopy (ESEM) allows the examination of hydrated and dried specimens without a conductive metal coating which could be advantageous in the imaging of biological and medical objects. The aim of this study was to assess the performance and benefits of wet-mode and low vacuum ESEM in comparison to high vacuum scanning electron microscopy (SEM) using the choroid plexus of chicken embryos as a model, an organ of the brain involved in the formation of cerebrospinal fluid in vertebrates. Specimens were fixed with or without heavy metals and examined directly or after critical point drying with or without metal coating. For wet mode ESEM freshly excised specimens without any pre-treatment were also examined. Conventional high vacuum SEM revealed the characteristic morphology of the choroid plexus cells at a high resolution and served as reference. With low vacuum ESEM of dried but uncoated samples the structure appeared well preserved but charging was a problem. It could be reduced by a short beam dwell time and averaging of images or by using the backscattered electron detector instead of the gaseous secondary electron detector. However, resolution was lower than with conventional SEM. Wet mode imaging was only possible with tissue that had been stabilized by fixation. Not all surface details (e.g. microvilli) could be visualized and other structures, like the cilia, were deformed. In summary, ESEM is an additional option for the imaging of bio-medical samples but it is problematic with regard to resolution and sample stability during imaging. Copyright © 2011 Elsevier GmbH. All rights reserved.

Nano-Optics for Chemical and Materials Characterization

NASA Astrophysics Data System (ADS)

Beversluis, Michael; Stranick, Stephan

2007-03-01

Light microscopy can provide non-destructive, real-time, three-dimensional imaging with chemically-specific contrast, but diffraction frequently limits the resolution to roughly 200 nm. Recently, structured illumination techniques have allowed fluorescence imaging to reach 50 nm resolution [1]. Since these fluorescence techniques were developed for use in microbiology, a key challenge is to take the resolution-enhancing features and apply them to contrast mechanisms like vibrational spectroscopy (e.g., Raman and CARS microscopy) that provide morphological and chemically specific imaging.. We are developing a new hybrid technique that combines the resolution enhancement of structured illumination microscopy with scanning techniques that can record hyperspectral images with 100 nm spatial resolution. We will show such superresolving images of semiconductor nanostructures and discuss the advantages and requirements for this technique. Referenence: 1. M. G. L. Gustafsson, P. Natl. Acad. Sci. USA 102, 13081-13086 (2005).

The application of laser scanning confocal microscopy to the examination of hairs and textile fibers: an initial investigation.

PubMed

Kirkbride, K Paul; Tridico, Silvana R

2010-02-25

An initial investigation of the application of laser scanning confocal microscopy to the examination of hairs and fibers has been conducted. This technique allows the production of virtual transverse and longitudinal cross-sectional images of a wide range of hairs and fibers. Special mounting techniques are not required; specimens that have been mounted for conventional microscopy require no further treatment. Unlike physical cross-sectioning, in which it is difficult to produce multiple cross-sections from a single hair or fiber and the process is destructive, confocal microscopy allows the examiner to image the cross-section at any point in the field of view along the hair or fiber and it is non-destructive. Confocal microscopy is a fluorescence-based technique. The images described in this article were collected using only the autofluorescence exhibited by the specimen (i.e. fluorescence staining was not necessary). Colorless fibers generally and hairs required excitation at 405 nm in order to stimulate useful autofluorescence; longer wavelength excitation was suitable for dyed fibers. Although confocal microscopy was found to be generally applicable to the generation virtual transverse cross-sections from a wide range of hairs and fibers, on some occasions the autofluorescence signal was attenuated by heavy pigmentation or the presence of an opaque medulla in hairs, and by heavy delustering or the presence of air-filled voids in the case of fibers. In these situations only partial cross-sections were obtained. 2009 Elsevier Ireland Ltd. All rights reserved.

Video-rate resonant scanning multiphoton microscopy

PubMed Central

Kirkpatrick, Nathaniel D.; Chung, Euiheon; Cook, Daniel C.; Han, Xiaoxing; Gruionu, Gabriel; Liao, Shan; Munn, Lance L.; Padera, Timothy P.; Fukumura, Dai; Jain, Rakesh K.

2013-01-01

The abnormal tumor microenvironment fuels tumor progression, metastasis, immune suppression, and treatment resistance. Over last several decades, developments in and applications of intravital microscopy have provided unprecedented insights into the dynamics of the tumor microenvironment. In particular, intravital multiphoton microscopy has revealed the abnormal structure and function of tumor-associated blood and lymphatic vessels, the role of aberrant tumor matrix in drug delivery, invasion and metastasis of tumor cells, the dynamics of immune cell trafficking to and within tumors, and gene expression in tumors. However, traditional multiphoton microscopy suffers from inherently slow imaging rates—only a few frames per second, thus unable to capture more rapid events such as blood flow, lymphatic flow, and cell movement within vessels. Here, we report the development and implementation of a video-rate multiphoton microscope (VR-MPLSM) based on resonant galvanometer mirror scanning that is capable of recording at 30 frames per second and acquiring intravital multispectral images. We show that the design of the system can be readily implemented and is adaptable to various experimental models. As examples, we demonstrate the utility of the system to directly measure flow within tumors, capture metastatic cancer cells moving within the brain vasculature and cells in lymphatic vessels, and image acute responses to changes in a vascular network. VR-MPLSM thus has the potential to further advance intravital imaging and provide new insight into the biology of the tumor microenvironment. PMID:24353926

Quantifying collagen orientation in breast tissue biopsies using SLIM (Conference Presentation)

NASA Astrophysics Data System (ADS)

Majeed, Hassaan; Okoro, Chukwuemeka; Balla, Andre; Toussaint, Kimani C.; Popescu, Gabriel

2017-02-01

Breast cancer is a major public health problem worldwide, being the most common type of cancer among women according to the World Health Organization (WHO). The WHO has further stressed the importance of an early determination of the disease course through prognostic markers. Recent studies have shown that the alignment of collagen fibers in tumor adjacent stroma correlate with poorer health outcomes in patients. Such studies have typically been carried out using Second-Harmonic Generation (SHG) microscopy. SHG images are very useful for quantifying collagen fiber orientation due their specificity to non-centrosymmetric structures in tissue, leading to high contrast in collagen rich areas. However, the imaging throughput in SHG microscopy is limited by its point scanning geometry. In this work, we show that SLIM, a wide-field high-throughput QPI technique, can be used to obtain the same information on collagen fiber orientation as is obtainable through SHG microscopy. We imaged a tissue microarray containing both benign and malignant cores using both SHG microscopy and SLIM. The cellular (non-collagenous) structures in the SLIM images were next segmented out using an algorithm developed in-house. Using the previously published Fourier Transform Second Harmonic Generation (FT-SHG) tool, the fiber orientations in SHG and segmented SLIM images were then quantified. The resulting histograms of fiber orientation angles showed that both SHG and SLIM generate similar measurements of collagen fiber orientation. The SLIM modality, however, can generate these results at much higher throughput due to its wide-field, whole-slide scanning capabilities.

Characterizing Surfaces of the Wide Bandgap Semiconductor Ilmenite with Scanning Probe Microcopies

NASA Technical Reports Server (NTRS)

Wilkins, R.; Powell, Kirk St. A.

1997-01-01

Ilmenite (FeTiO3) is a wide bandgap semiconductor with an energy gap of about 2.5eV. Initial radiation studies indicate that ilmenite has properties suited for radiation tolerant applications, as well as a variety of other electronic applications. Two scanning probe microscopy methods have been used to characterize the surface of samples taken from Czochralski grown single crystals. The two methods, atomic force microscopy (AFM) and scanning tunneling microscopy (STM), are based on different physical principles and therefore provide different information about the samples. AFM provides a direct, three-dimensional image of the surface of the samples, while STM give a convolution of topographic and electronic properties of the surface. We will discuss the differences between the methods and present preliminary data of each method for ilmenite samples.

Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy

PubMed Central

Shimura, Mari; Shindou, Hideo; Szyrwiel, Lukasz; Tokuoka, Suzumi M.; Hamano, Fumie; Matsuyama, Satoshi; Okamoto, Mayumi; Matsunaga, Akihiro; Kita, Yoshihiro; Ishizaka, Yukihito; Yamauchi, Kazuto; Kohmura, Yoshiki; Lobinski, Ryszard; Shimizu, Isao; Shimizu, Takao

2016-01-01

Fatty acids are taken up by cells and incorporated into complex lipids such as neutral lipids and glycerophospholipids. Glycerophospholipids are major constituents of cellular membranes. More than 1000 molecular species of glycerophospholipids differ in their polar head groups and fatty acid compositions. They are related to cellular functions and diseases and have been well analyzed by mass spectrometry. However, intracellular imaging of fatty acids and glycerophospholipids has not been successful due to insufficient resolution using conventional methods. Here, we developed a method for labeling fatty acids with bromine (Br) and applied scanning X-ray fluorescence microscopy (SXFM) to obtain intracellular Br mapping data with submicrometer resolution. Mass spectrometry showed that cells took up Br-labeled fatty acids and metabolized them mainly into glycerophospholipids in CHO cells. Most Br signals observed by SXFM were in the perinuclear region. Higher resolution revealed a spot-like distribution of Br in the cytoplasm. The current method enabled successful visualization of intracellular Br-labeled fatty acids. Single-element labeling combined with SXFM technology facilitates the intracellular imaging of fatty acids, which provides a new tool to determine dynamic changes in fatty acids and their derivatives at the single-cell level.—Shimura, M., Shindou, H., Szyrwiel, L., Tokuoka, S. M., Hamano, F., Matsuyama, S., Okamoto, M., Matsunaga, A., Kita, Y., Ishizaka, Y., Yamauchi, K., Kohmura, Y., Lobinski, R., Shimizu, I., Shimizu, T. Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy. PMID:27601443

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.

The quest for four-dimensional imaging in plant cell biology: it's just a matter of time

PubMed Central

Domozych, David S.

2012-01-01

Background Analysis of plant cell dynamics over time, or four-dimensional imaging (4-DI), represents a major goal of plant science. The ability to resolve structures in the third dimension within the cell or tissue during developmental events or in response to environmental or experimental stresses (i.e. 4-DI) is critical to our understanding of gene expression, post-expression modulations of macromolecules and sub-cellular system interactions. Scope Microscopy-based technologies have been profoundly integral to this type of investigation, and new and refined microscopy technologies now allow for the visualization of cell dynamics with unprecedented resolution, contrast and experimental versatility. However, certain realities of light and electron microscopy, choice of specimen and specimen preparation techniques limit the scope of readily attaining 4-DI. Today, the plant microscopist must use a combinatorial strategy whereby multiple microscopy-based investigations are used. Modern fluorescence, confocal laser scanning, transmission electron and scanning electron microscopy provide effective conduits for synthesizing data detailing live cell dynamics and highly resolved snapshots of specific cell structures that will ultimately lead to 4-DI. This review provides a synopsis of such technologies available. PMID:22628381

Transmission/Scanning Transmission Electron Microscopy | Materials Science

Science.gov Websites

imaging such as high resolution TEM. Transmission electron diffraction patterns help to determine the microstructure of a material and its defects. Phase-contrast imaging or high-resolution (HR) TEM imaging gives high scattering angle can be collected to form high-resolution, chemically sensitive, atomic number (Z

Ultrahigh speed en face OCT capsule for endoscopic imaging

PubMed Central

Liang, Kaicheng; Traverso, Giovanni; Lee, Hsiang-Chieh; Ahsen, Osman Oguz; Wang, Zhao; Potsaid, Benjamin; Giacomelli, Michael; Jayaraman, Vijaysekhar; Barman, Ross; Cable, Alex; Mashimo, Hiroshi; Langer, Robert; Fujimoto, James G.

2015-01-01

Depth resolved and en face OCT visualization in vivo may have important clinical applications in endoscopy. We demonstrate a high speed, two-dimensional (2D) distal scanning capsule with a micromotor for fast rotary scanning and a pneumatic actuator for precision longitudinal scanning. Longitudinal position measurement and image registration were performed by optical tracking of the pneumatic scanner. The 2D scanning device enables high resolution imaging over a small field of view and is suitable for OCT as well as other scanning microscopies. Large field of view imaging for screening or surveillance applications can also be achieved by proximally pulling back or advancing the capsule while scanning the distal high-speed micromotor. Circumferential en face OCT was demonstrated in living swine at 250 Hz frame rate and 1 MHz A-scan rate using a MEMS tunable VCSEL light source at 1300 nm. Cross-sectional and en face OCT views of the upper and lower gastrointestinal tract were generated with precision distal pneumatic longitudinal actuation as well as proximal manual longitudinal actuation. These devices could enable clinical studies either as an adjunct to endoscopy, attached to an endoscope, or as a swallowed tethered capsule for non-endoscopic imaging without sedation. The combination of ultrahigh speed imaging and distal scanning capsule technology could enable both screening and surveillance applications. PMID:25909001

Ultrahigh speed en face OCT capsule for endoscopic imaging.

PubMed

Liang, Kaicheng; Traverso, Giovanni; Lee, Hsiang-Chieh; Ahsen, Osman Oguz; Wang, Zhao; Potsaid, Benjamin; Giacomelli, Michael; Jayaraman, Vijaysekhar; Barman, Ross; Cable, Alex; Mashimo, Hiroshi; Langer, Robert; Fujimoto, James G

2015-04-01

Depth resolved and en face OCT visualization in vivo may have important clinical applications in endoscopy. We demonstrate a high speed, two-dimensional (2D) distal scanning capsule with a micromotor for fast rotary scanning and a pneumatic actuator for precision longitudinal scanning. Longitudinal position measurement and image registration were performed by optical tracking of the pneumatic scanner. The 2D scanning device enables high resolution imaging over a small field of view and is suitable for OCT as well as other scanning microscopies. Large field of view imaging for screening or surveillance applications can also be achieved by proximally pulling back or advancing the capsule while scanning the distal high-speed micromotor. Circumferential en face OCT was demonstrated in living swine at 250 Hz frame rate and 1 MHz A-scan rate using a MEMS tunable VCSEL light source at 1300 nm. Cross-sectional and en face OCT views of the upper and lower gastrointestinal tract were generated with precision distal pneumatic longitudinal actuation as well as proximal manual longitudinal actuation. These devices could enable clinical studies either as an adjunct to endoscopy, attached to an endoscope, or as a swallowed tethered capsule for non-endoscopic imaging without sedation. The combination of ultrahigh speed imaging and distal scanning capsule technology could enable both screening and surveillance applications.

Correlative two-photon and serial block face scanning electron microscopy in neuronal tissue using 3D near-infrared branding maps.

PubMed

Lees, Robert M; Peddie, Christopher J; Collinson, Lucy M; Ashby, Michael C; Verkade, Paul

2017-01-01

Linking cellular structure and function has always been a key goal of microscopy, but obtaining high resolution spatial and temporal information from the same specimen is a fundamental challenge. Two-photon (2P) microscopy allows imaging deep inside intact tissue, bringing great insight into the structural and functional dynamics of cells in their physiological environment. At the nanoscale, the complex ultrastructure of a cell's environment in tissue can be reconstructed in three dimensions (3D) using serial block face scanning electron microscopy (SBF-SEM). This provides a snapshot of high resolution structural information pertaining to the shape, organization, and localization of multiple subcellular structures at the same time. The pairing of these two imaging modalities in the same specimen provides key information to relate cellular dynamics to the ultrastructural environment. Until recently, approaches to relocate a region of interest (ROI) in tissue from 2P microscopy for SBF-SEM have been inefficient or unreliable. However, near-infrared branding (NIRB) overcomes this by using the laser from a multiphoton microscope to create fiducial markers for accurate correlation of 2P and electron microscopy (EM) imaging volumes. The process is quick and can be user defined for each sample. Here, to increase the efficiency of ROI relocation, multiple NIRB marks are used in 3D to target ultramicrotomy. A workflow is described and discussed to obtain a data set for 3D correlated light and electron microscopy, using three different preparations of brain tissue as examples. Copyright © 2017 Elsevier Inc. All rights reserved.

Preparation of scanning tunneling microscopy tips using pulsed alternating current etching

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

Valencia, Victor A.; Thaker, Avesh A.; Derouin, Jonathan

An electrochemical method using pulsed alternating current etching (PACE) to produce atomically sharp scanning tunneling microscopy (STM) tips is presented. An Arduino Uno microcontroller was used to control the number and duration of the alternating current (AC) pulses, allowing for ready optimization of the procedures for both Pt:Ir and W tips using a single apparatus. W tips prepared using constant and pulsed AC power were compared. Tips fashioned using PACE were sharper than those etched with continuous AC power alone. Pt:Ir tips were prepared with an initial coarse etching stage using continuous AC power followed by fine etching using PACE.more » The number and potential of the finishing AC pulses was varied and scanning electron microscope imaging was used to compare the results. Finally, tip quality using the optimized procedures was verified by UHV-STM imaging. With PACE, at least 70% of the W tips and 80% of the Pt:Ir tips were of sufficiently high quality to obtain atomically resolved images of HOPG or Ni(111)« less

Fast spectral coherent anti-Stokes Raman scattering microscopy with high-speed tunable picosecond laser.

PubMed

Cahyadi, Harsono; Iwatsuka, Junichi; Minamikawa, Takeo; Niioka, Hirohiko; Araki, Tsutomu; Hashimoto, Mamoru

2013-09-01

We develop a coherent anti-Stokes Raman scattering (CARS) microscopy system equipped with a tunable picosecond laser for high-speed wavelength scanning. An acousto-optic tunable filter (AOTF) is integrated in the laser cavity to enable wavelength scanning by varying the radio frequency waves applied to the AOTF crystal. An end mirror attached on a piezoelectric actuator and a pair of parallel plates driven by galvanometer motors are also introduced into the cavity to compensate for changes in the cavity length during wavelength scanning to allow synchronization with another picosecond laser. We demonstrate fast spectral imaging of 3T3-L1 adipocytes every 5  cm-1 in the Raman spectral region around 2850  cm-1 with an image acquisition time of 120 ms. We also demonstrate fast switching of Raman shifts between 2100 and 2850  cm-1, corresponding to CD2 symmetric stretching and CH2 symmetric stretching vibrations, respectively. The fast-switching CARS images reveal different locations of recrystallized deuterated and nondeuterated stearic acid.

Fast imaging with inelastically scattered electrons by off-axis chromatic confocal electron microscopy.

PubMed

Zheng, Changlin; Zhu, Ye; Lazar, Sorin; Etheridge, Joanne

2014-04-25

We introduce off-axis chromatic scanning confocal electron microscopy, a technique for fast mapping of inelastically scattered electrons in a scanning transmission electron microscope without a spectrometer. The off-axis confocal mode enables the inelastically scattered electrons to be chromatically dispersed both parallel and perpendicular to the optic axis. This enables electrons with different energy losses to be separated and detected in the image plane, enabling efficient energy filtering in a confocal mode with an integrating detector. We describe the experimental configuration and demonstrate the method with nanoscale core-loss chemical mapping of silver (M4,5) in an aluminium-silver alloy and atomic scale imaging of the low intensity core-loss La (M4,5@840  eV) signal in LaB6. Scan rates up to 2 orders of magnitude faster than conventional methods were used, enabling a corresponding reduction in radiation dose and increase in the field of view. If coupled with the enhanced depth and lateral resolution of the incoherent confocal configuration, this offers an approach for nanoscale three-dimensional chemical mapping.

Joint denoising and distortion correction of atomic scale scanning transmission electron microscopy images

NASA Astrophysics Data System (ADS)

Berkels, Benjamin; Wirth, Benedikt

2017-09-01

Nowadays, modern electron microscopes deliver images at atomic scale. The precise atomic structure encodes information about material properties. Thus, an important ingredient in the image analysis is to locate the centers of the atoms shown in micrographs as precisely as possible. Here, we consider scanning transmission electron microscopy (STEM), which acquires data in a rastering pattern, pixel by pixel. Due to this rastering combined with the magnification to atomic scale, movements of the specimen even at the nanometer scale lead to random image distortions that make precise atom localization difficult. Given a series of STEM images, we derive a Bayesian method that jointly estimates the distortion in each image and reconstructs the underlying atomic grid of the material by fitting the atom bumps with suitable bump functions. The resulting highly non-convex minimization problems are solved numerically with a trust region approach. Existence of minimizers and the model behavior for faster and faster rastering are investigated using variational techniques. The performance of the method is finally evaluated on both synthetic and real experimental data.

Application of gold nanoparticles as contrast agents in confocal laser scanning microscopy

NASA Astrophysics Data System (ADS)

Lemelle, A.; Veksler, B.; Kozhevnikov, I. S.; Akchurin, G. G.; Piletsky, S. A.; Meglinski, I.

2009-01-01

Confocal laser scanning microscopy (CLSM) is a modern high-resolution optical technique providing detailed image of tissue structure with high (down to microns) spatial resolution. Aiming at a concurrent improvement of imaging depth and image quality the CLSM requires the use of contrast agents. Commonly employed fluorescent contrast agents, such as fluorescent dyes and proteins, suffer from toxicity, photo-bleaching and overlapping with the tissues autofluorescence. Gold nanoparticles are potentially highly attractive to be applied as a contrast agent since they are not subject to photo-bleaching and can target biochemical cells markers associated with the specific diseases. In current report we consider the applicability of gold nano-spheres as a contrast agent to enhance quality of CLSM images of skin tissues in vitro versus the application of optical clearing agent, such as glycerol. The enhancement of CLSM image contrast was observed with an application of gold nano-spheres diffused within the skin tissues. We show that optical clearing agents such as a glycerol provide better CLSM image contrast than gold nano-spheres.

A novel method for enhancing the lateral resolution and image SNR in confocal microscopy

NASA Astrophysics Data System (ADS)

Chen, Youhua; Zhu, Dazhao; Fang, Yue; Kuang, Cuifang; Liu, Xu

2017-12-01

There is always a tradeoff between the resolution and the signal-to-noise ratio (SNR) in confocal microscopy. In particular, the pinhole size is very important for maintaining a balance between them. In this paper, we propose a method for improving the lateral resolution and image SNR in confocal microscopy without making any changes to the hardware. By using the fluorescence emission difference (FED) approach, we divide the images acquired by different pinhole sizes into one image acquired by the central pinhole and several images acquired by ring-shaped pinholes. Then, they are added together with the deconvolution method. Simulation and experimental results for fluorescent particles and cells show that our method can achieve a far better resolution than a large pinhole and a higher SNR than a small pinhole. Moreover, our method can improve the performance of classic confocal laser scanning microscopy (CLSM) to a certain extent, especially CLSM with a continuously variable pinhole.

Enhanced resolution imaging of ultrathin ZnO layers on Ag(111) by multiple hydrogen molecules in a scanning tunneling microscope junction

NASA Astrophysics Data System (ADS)

Liu, Shuyi; Shiotari, Akitoshi; Baugh, Delroy; Wolf, Martin; Kumagai, Takashi

2018-05-01

Molecular hydrogen in a scanning tunneling microscope (STM) junction has been found to enhance the lateral spatial resolution of the STM imaging, referred to as scanning tunneling hydrogen microscopy (STHM). Here we report atomic resolution imaging of 2- and 3-monolayer (ML) thick ZnO layers epitaxially grown on Ag(111) using STHM. The enhanced resolution can be obtained at a relatively large tip to surface distance and resolves a more defective structure exhibiting dislocation defects for 3-ML-thick ZnO than for 2 ML. In order to elucidate the enhanced imaging mechanism, the electric and mechanical properties of the hydrogen molecular junction (HMJ) are investigated by a combination of STM and atomic force microscopy. It is found that the HMJ shows multiple kinklike features in the tip to surface distance dependence of the conductance and frequency shift curves, which are absent in a hydrogen-free junction. Based on a simple modeling, we propose that the junction contains several hydrogen molecules and sequential squeezing of the molecules out of the junction results in the kinklike features in the conductance and frequency shift curves. The model also qualitatively reproduces the enhanced resolution image of the ZnO films.

Electron Energy Loss Spectral Imaging of TiC Formed by Supernovae: A Scanning Transmission Electron Microscopy Study of Grain Formation and Alteration Mechanisms

NASA Astrophysics Data System (ADS)

Daulton, T. L.; Bernatowicz, T. J.; Croat, T. K.

2012-03-01

Micrometer-sized spherules of graphite formed by supernovae contain numerous TiC and Fe-Ni subgrains. These subgrains often have disordered surface rims. The mechanism(s) of rim formation on these subgrains is studied by transmission electron microscopy.

Morphological development of cellulose fibrils of a bleached eucalyptus pulp by mechanical fibrillation

Treesearch

Q.Q. Wang; J.Y. Zhu; R. Gleisner; T.A. Kuster; U. Baxa; S.E. McNeil

2012-01-01

This study reports the production of cellulose nanofibrils (CNF) from a bleached eucalyptus pulp using a commercial stone grinder. Scanning electronic microscopy and transmission electronic microscopy imaging were used to reveal morphological development of CNF at micro and nano scales, respectively. Two major structures were identified (1) highly kinked, naturally...

New modes of electron microscopy for materials science enabled by fast direct electron detectors

NASA Astrophysics Data System (ADS)

Minor, Andrew

There is an ongoing revolution in the development of electron detector technology that has enabled modes of electron microscopy imaging that had only before been theorized. The age of electron microscopy as a tool for imaging is quickly giving way to a new frontier of multidimensional datasets to be mined. These improvements in electron detection have enabled cryo-electron microscopy to resolve the three-dimensional structures of non-crystalized proteins, revolutionizing structural biology. In the physical sciences direct electron detectors has enabled four-dimensional reciprocal space maps of materials at atomic resolution, providing all the structural information about nanoscale materials in one experiment. This talk will highlight the impact of direct electron detectors for materials science, including a new method of scanning nanobeam diffraction. With faster detectors we can take a series of 2D diffraction patterns at each position in a 2D STEM raster scan resulting in a four-dimensional data set. For thin film analysis, direct electron detectors hold the potential to enable strain, polarization, composition and electrical field mapping over relatively large fields of view, all from a single experiment.

Probe-based confocal laser endomicroscopy (pCLE) - a new imaging technique for in situ localization of spermatozoa.

PubMed

Trottmann, Matthias; Stepp, Herbert; Sroka, Ronald; Heide, Michael; Liedl, Bernhard; Reese, Sven; Becker, Armin J; Stief, Christian G; Kölle, Sabine

2015-05-01

In azoospermic patients, spermatozoa are routinely obtained by testicular sperm extraction (TESE). However, success rates of this technique are moderate, because the site of excision of testicular tissue is determined arbitrarily. Therefore the aim of this study was to establish probe-based laser endomicroscopy (pCLE) a noval biomedical imaging technique, which provides the opportunity of non-invasive, real-time visualisation of tissue at histological resolution. Using pCLE we clearly visualized longitudinal and horizontal views of the tubuli seminiferi contorti and localized vital spermatozoa. Obtained images and real-time videos were subsequently compared with confocal laser scanning microscopy (CLSM) of spermatozoa and tissues, respectively. Comparative visualization of single native Confocal laser scanning microscopy (CLSM, left) and probe-based laser endomicroscopy (pCLE, right) using Pro Flex(TM) UltraMini O after staining with acriflavine. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cryo-scanning transmission electron tomography of vitrified cells.

PubMed

Wolf, Sharon Grayer; Houben, Lothar; Elbaum, Michael

2014-04-01

Cryo-electron tomography (CET) of fully hydrated, vitrified biological specimens has emerged as a vital tool for biological research. For cellular studies, the conventional imaging modality of transmission electron microscopy places stringent constraints on sample thickness because of its dependence on phase coherence for contrast generation. Here we demonstrate the feasibility of using scanning transmission electron microscopy for cryo-tomography of unstained vitrified specimens (CSTET). We compare CSTET and CET for the imaging of whole bacteria and human tissue culture cells, finding favorable contrast and detail in the CSTET reconstructions. Particularly at high sample tilts, the CSTET signals contain more informative data than energy-filtered CET phase contrast images, resulting in improved depth resolution. Careful control over dose delivery permits relatively high cumulative exposures before the onset of observable beam damage. The increase in acceptable specimen thickness broadens the applicability of electron cryo-tomography.

Quantitative Transmission Electron Microscopy of Nanoparticles and Thin-Film Formation in Electroless Metallization of Polymeric Surfaces

NASA Astrophysics Data System (ADS)

Dutta, Aniruddha; Heinrich, Helge; Kuebler, Stephen; Grabill, Chris; Bhattacharya, Aniket

2011-03-01

Gold nanoparticles(Au-NPs) act as nucleation sites for electroless deposition of silver on functionalized SU8 polymeric surfaces. Here we report the nanoscale morphology of Au and Ag nanoparticles as studied by Transmission Electron Microscopy (TEM). Scanning TEM with a high-angle annular dark-field detector is used to obtain atomic number contrast. From the intensity-calibrated plan-view scanning TEM images we determine the mean thickness and the volume distribution of the Au-NPs on the surface of the functionalized polymer. We also report the height and the radius distribution of the gold nanoparticles obtained from STEM images taking into consideration the experimental errors. The cross sectional TEM images yield the density and the average distance of the Au and Ag nanoparticles on the surface of the polymer. Supported by grant NSF, Chemistry Division.

Oxidation-state sensitive imaging of cerium dioxide by atomic-resolution low-angle annular dark field scanning transmission electron microscopy.

PubMed

Johnston-Peck, Aaron C; Winterstein, Jonathan P; Roberts, Alan D; DuChene, Joseph S; Qian, Kun; Sweeny, Brendan C; Wei, Wei David; Sharma, Renu; Stach, Eric A; Herzing, Andrew A

2016-03-01

Low-angle annular dark field (LAADF) scanning transmission electron microscopy (STEM) imaging is presented as a method that is sensitive to the oxidation state of cerium ions in CeO2 nanoparticles. This relationship was validated through electron energy loss spectroscopy (EELS), in situ measurements, as well as multislice image simulations. Static displacements caused by the increased ionic radius of Ce(3+) influence the electron channeling process and increase electron scattering to low angles while reducing scatter to high angles. This process manifests itself by reducing the high-angle annular dark field (HAADF) signal intensity while increasing the LAADF signal intensity in close proximity to Ce(3+) ions. This technique can supplement STEM-EELS and in so doing, relax the experimental challenges associated with acquiring oxidation state information at high spatial resolutions. Published by Elsevier B.V.

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.

Characterization of konjac glucomannan-ethyl cellulose film formation via microscopy.

PubMed

Xiao, Man; Wan, Li; Corke, Harold; Yan, Wenli; Ni, Xuewen; Fang, Yapeng; Jiang, Fatang

2016-04-01

Konjac glucomannan-ethyl cellulose (KGM-EC, 7:3, w/w) blended film shows good mechanical and moisture resistance properties. To better understand the basis for the KGM-EC film formation, optical microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) were used to observe the formation of the film from emulsion. Optical microscopy images showed that EC oil droplets were homogeneously dispersed in KGM water phase without obviously coalescence throughout the entire drying process. SEM images showed the surface and cross-sectional structures of samples maintained continuous and homogeneous appearance from the emulsion to dried film. AFM images indicated that KGM molecules entangled EC molecules in the emulsion. Interactions between KGM and EC improved the stability of KGM-EC emulsion, and contributed to uniformed structures of film formation. Based on these output information, a schematic model was built to elucidate KGM-EC film-forming process. Copyright © 2015 Elsevier B.V. All rights reserved.

Multidirectional Image Sensing for Microscopy Based on a Rotatable Robot.

PubMed

Shen, Yajing; Wan, Wenfeng; Zhang, Lijun; Yong, Li; Lu, Haojian; Ding, Weili

2015-12-15

Image sensing at a small scale is essentially important in many fields, including microsample observation, defect inspection, material characterization and so on. However, nowadays, multi-directional micro object imaging is still very challenging due to the limited field of view (FOV) of microscopes. This paper reports a novel approach for multi-directional image sensing in microscopes by developing a rotatable robot. First, a robot with endless rotation ability is designed and integrated with the microscope. Then, the micro object is aligned to the rotation axis of the robot automatically based on the proposed forward-backward alignment strategy. After that, multi-directional images of the sample can be obtained by rotating the robot within one revolution under the microscope. To demonstrate the versatility of this approach, we view various types of micro samples from multiple directions in both optical microscopy and scanning electron microscopy, and panoramic images of the samples are processed as well. The proposed method paves a new way for the microscopy image sensing, and we believe it could have significant impact in many fields, especially for sample detection, manipulation and characterization at a small scale.

Carbon nanotube scanning probe for imaging in aqueous environment

NASA Technical Reports Server (NTRS)

Stevens, Ramsey M.; Nguyen, Cattien V.; Meyyappan, M.

2004-01-01

Carbon nanotubes (CNTs) used as a probe for scanning probe microscopy has become one of the many potential usages of CNTs that is finding real applications in scientific research and industrial communities. It has been proposed that the unique mechanical buckling properties of the CNT would lessen the imaging force exerted on the sample and, thus, make CNT scanning probes ideal for imaging soft materials, including biological samples in liquid environments. The hydrophobic nature of the CNT graphitic sidewall is clearly chemically incompatible with the aqueous solution requirements in some biological imaging applications. In this paper, we present electron micrograph results demonstrating the instability of CNT scanning probes when submerged in aqueous solution. Moreover, we also introduce a novel approach to resolve this chemical incompatibility problem. By coating the CNT probe with ethylenediamine, thus rendering the CNT probe less hydrophobic, we demonstrate the liquid imaging capability of treated CNT probes. Experimental data for imaging in aqueous solutions are presented, which include an ultrathin Ir film and DNA molecules on a mica surface.

Through-focus scanning optical microscopy (TSOM) with adaptive optics

NASA Astrophysics Data System (ADS)

Lee, Jun Ho; Park, Gyunam; Jeong, Junhee; Park, Chris

2018-03-01

Through-focus optical microscopy (TSOM) with nanometer-scale lateral and vertical sensitivity levels matching those of scanning electron microscopy has been demonstrated to be useful both for 3D inspections and metrology assessments. In 2014, funded by two private companies (Nextin/Samsung Electronics) and the Korea Evaluation Institute of Industrial Technology (KEIT), a research team from four universities in South Korea set out to investigate core technologies for developing in-line TSOM inspection and metrology tools, with the respective teams focusing on optics implementation, defect inspection, computer simulation and high-speed metrology matching. We initially confirmed the reported validity of the TSOM operation through a computer simulation, after which we implemented the TSOM operation by throughfocus scanning of existing UV (355nm) and IR (800nm) inspection tools. These tools have an identical sampling distance of 150 nm but have different resolving distances (310 and 810 nm, respectively). We initially experienced some improvement in the defect inspection sensitivity level over TSV (through-silicon via) samples with 6.6 μm diameters. However, during the experiment, we noted sensitivity and instability issues when attempting to acquire TSOM images. As TSOM 3D information is indirectly extracted by differentiating a target TSOM image from reference TSOM images, any instability or mismatch in imaging conditions can result in measurement errors. As a remedy to such a situation, we proposed the application of adaptive optics to the TSOM operation and developed a closed-loop system with a tip/tilt mirror and a Shack-Hartmann sensor on an optical bench. We were able to keep the plane position within in RMS 0.4 pixel by actively compensating for any position instability which arose during the TSOM scanning process along the optical axis. Currently, we are also developing another TSOM tool with a deformable mirror instead of a tip/tilt mirror, in which case we will not require any mechanical scanning.

Advanced techniques for in situ analysis of the biofilm matrix (structure, composition, dynamics) by means of laser scanning microscopy.

PubMed

Neu, Thomas R; Lawrence, John R

2014-01-01

The extracellular constituents in bioaggregates and biofilms can be imaged four dimensionally by using laser scanning microscopy. In this protocol we provide guidance on how to examine the various extracellular compartments in between microbial cells and communities associated with interfaces. The current options for fluorescence staining of matrix compounds and extracellular microhabitats are presented. Furthermore, practical aspects are discussed and useful notes are added. The chapter ends with a brief introduction to other approaches for EPS analysis and an outlook for future needs.

Monolithically Integrated, Mechanically Resilient Carbon-Based Probes for Scanning Probe Microscopy

NASA Technical Reports Server (NTRS)

Kaul, Anupama B.; Megerian, Krikor G.; Jennings, Andrew T.; Greer, Julia R.

2010-01-01

Scanning probe microscopy (SPM) is an important tool for performing measurements at the nanoscale in imaging bacteria or proteins in biology, as well as in the electronics industry. An essential element of SPM is a sharp, stable tip that possesses a small radius of curvature to enhance spatial resolution. Existing techniques for forming such tips are not ideal. High-aspect-ratio, monolithically integrated, as-grown carbon nanofibers (CNFs) have been formed that show promise for SPM applications by overcoming the limitations present in wet chemical and separate substrate etching processes.

Thermally oxidized Inconel 600 and 690 nickel-based alloys characterizations by combination of global photoelectrochemistry and local near-field microscopy techniques (STM, STS, AFM, SKPFM)

NASA Astrophysics Data System (ADS)

Mechehoud, F.; Benaioun, N. E.; Hakiki, N. E.; Khelil, A.; Simon, L.; Bubendorff, J. L.

2018-03-01

Thermally oxidized nickel-based alloys are studied by scanning tunnelling microscopy (STM), scanning tunnelling spectroscopy (STS), atomic force microscopy (AFM), scanning kelvin probe force microscopy (SKPFM) and photoelectro-chemical techniques as a function of oxidation time at a fixed temperature of 623 K. By photoelectrochemistry measurements we identify the formation of three oxides NiO, Fe2O3, Cr2O3 and determine the corresponding gap values. We use these values as parameter for imaging the surface at high bias voltage by STM allowing the spatial localization and identification of both NiO, Fe2O3 oxide phases using STS measurements. Associated to Kelvin probe measurements we show also that STS allow to distinguished NiO from Cr2O3 and confirm that the Cr2O3 is not visible at the surface and localized at the oxide/steel interface.

Epidermal growth factor receptor subunit locations determined in hydrated cells with environmental scanning electron microscopy.

PubMed

Peckys, Diana B; Baudoin, Jean-Pierre; Eder, Magdalena; Werner, Ulf; de Jonge, Niels

2013-01-01

Imaging single epidermal growth factor receptors (EGFR) in intact cells is presently limited by the available microscopy methods. Environmental scanning electron microscopy (ESEM) of whole cells in hydrated state in combination with specific labeling with gold nanoparticles was used to localize activated EGFRs in the plasma membranes of COS7 and A549 cells. The use of a scanning transmission electron microscopy (STEM) detector yielded a spatial resolution of 3 nm, sufficient to identify the locations of individual EGFR dimer subunits. The sizes and distribution of dimers and higher order clusters of EGFRs were determined. The distance between labels bound to dimers amounted to 19 nm, consistent with a molecular model. A fraction of the EGFRs was found in higher order clusters with sizes ranging from 32-56 nm. ESEM can be used for quantitative whole cell screening studies of membrane receptors, and for the study of nanoparticle-cell interactions in general.

Epidermal growth factor receptor subunit locations determined in hydrated cells with environmental scanning electron microscopy

PubMed Central

Peckys, Diana B.; Baudoin, Jean-Pierre; Eder, Magdalena; Werner, Ulf; de Jonge, Niels

2013-01-01

Imaging single epidermal growth factor receptors (EGFR) in intact cells is presently limited by the available microscopy methods. Environmental scanning electron microscopy (ESEM) of whole cells in hydrated state in combination with specific labeling with gold nanoparticles was used to localize activated EGFRs in the plasma membranes of COS7 and A549 cells. The use of a scanning transmission electron microscopy (STEM) detector yielded a spatial resolution of 3 nm, sufficient to identify the locations of individual EGFR dimer subunits. The sizes and distribution of dimers and higher order clusters of EGFRs were determined. The distance between labels bound to dimers amounted to 19 nm, consistent with a molecular model. A fraction of the EGFRs was found in higher order clusters with sizes ranging from 32–56 nm. ESEM can be used for quantitative whole cell screening studies of membrane receptors, and for the study of nanoparticle-cell interactions in general. PMID:24022088

Three-dimensional imaging of porous media using confocal laser scanning microscopy.

PubMed

Shah, S M; Crawshaw, J P; Boek, E S

2017-02-01

In the last decade, imaging techniques capable of reconstructing three-dimensional (3-D) pore-scale model have played a pivotal role in the study of fluid flow through complex porous media. In this study, we present advances in the application of confocal laser scanning microscopy (CLSM) to image, reconstruct and characterize complex porous geological materials with hydrocarbon reservoir and CO 2 storage potential. CLSM has a unique capability of producing 3-D thin optical sections of a material, with a wide field of view and submicron resolution in the lateral and axial planes. However, CLSM is limited in the depth (z-dimension) that can be imaged in porous materials. In this study, we introduce a 'grind and slice' technique to overcome this limitation. We discuss the practical and technical aspects of the confocal imaging technique with application to complex rock samples including Mt. Gambier and Ketton carbonates. We then describe the complete workflow of image processing to filtering and segmenting the raw 3-D confocal volumetric data into pores and grains. Finally, we use the resulting 3-D pore-scale binarized confocal data obtained to quantitatively determine petrophysical pore-scale properties such as total porosity, macro- and microporosity and single-phase permeability using lattice Boltzmann (LB) simulations, validated by experiments. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

A scanning transmission electron microscopy approach to analyzing large volumes of tissue to detect nanoparticles.

PubMed

Kempen, Paul J; Thakor, Avnesh S; Zavaleta, Cristina; Gambhir, Sanjiv S; Sinclair, Robert

2013-10-01

The use of nanoparticles for the diagnosis and treatment of cancer requires the complete characterization of their toxicity, including accurately locating them within biological tissues. Owing to their size, traditional light microscopy techniques are unable to resolve them. Transmission electron microscopy provides the necessary spatial resolution to image individual nanoparticles in tissue, but is severely limited by the very small analysis volume, usually on the order of tens of cubic microns. In this work, we developed a scanning transmission electron microscopy (STEM) approach to analyze large volumes of tissue for the presence of polyethylene glycol-coated Raman-active-silica-gold-nanoparticles (PEG-R-Si-Au-NPs). This approach utilizes the simultaneous bright and dark field imaging capabilities of STEM along with careful control of the image contrast settings to readily identify PEG-R-Si-Au-NPs in mouse liver tissue without the need for additional time-consuming analytical characterization. We utilized this technique to analyze 243,000 mm³ of mouse liver tissue for the presence of PEG-R-Si-Au-NPs. Nanoparticles injected into the mice intravenously via the tail vein accumulated in the liver, whereas those injected intrarectally did not, indicating that they remain in the colon and do not pass through the colon wall into the systemic circulation.

Weak-beam scanning transmission electron microscopy for quantitative dislocation density measurement in steels.

PubMed

Yoshida, Kenta; Shimodaira, Masaki; Toyama, Takeshi; Shimizu, Yasuo; Inoue, Koji; Yoshiie, Toshimasa; Milan, Konstantinovic J; Gerard, Robert; Nagai, Yasuyoshi

2017-04-01

To evaluate dislocations induced by neutron irradiation, we developed a weak-beam scanning transmission electron microscopy (WB-STEM) system by installing a novel beam selector, an annular detector, a high-speed CCD camera and an imaging filter in the camera chamber of a spherical aberration-corrected transmission electron microscope. The capabilities of the WB-STEM with respect to wide-view imaging, real-time diffraction monitoring and multi-contrast imaging are demonstrated using typical reactor pressure vessel steel that had been used in an European nuclear reactor for 30 years as a surveillance test piece with a fluence of 1.09 × 1020 neutrons cm-2. The quantitatively measured size distribution (average loop size = 3.6 ± 2.1 nm), number density of the dislocation loops (3.6 × 1022 m-3) and dislocation density (7.8 × 1013 m m-3) were carefully compared with the values obtained via conventional weak-beam transmission electron microscopy studies. In addition, cluster analysis using atom probe tomography (APT) further demonstrated the potential of the WB-STEM for correlative electron tomography/APT experiments. © The Author 2017. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A Scanning Transmission Electron Microscopy (STEM) Approach to Analyzing Large Volumes of Tissue to Detect Nanoparticles

PubMed Central

Kempen, Paul J.; Thakor, Avnesh S.; Zavaleta, Cristina; Gambhir, Sanjiv S.; Sinclair, Robert

2013-01-01

The use of nanoparticles for the diagnosis and treatment of cancer requires the complete characterization of their toxicity, including accurately locating them within biological tissues. Owing to their size, traditional light microscopy techniques are unable to resolve them. Transmission electron microscopy provides the necessary spatial resolution to image individual nanoparticles in tissue but is severely limited by the very small analysis volume, usually on the order of tens of cubic microns. In this work we developed a scanning transmission electron microscopy (STEM) approach to analyze large volumes of tissue for the presence of polyethylene glycol coated Raman-active-silica-gold-nanoparticles (PEG-R-Si-Au-NPs). This approach utilizes the simultaneous bright and dark field imaging capabilities of STEM along with careful control of the image contrast settings to readily identify PEG-R-Si-Au-NPs in mouse liver tissue without the need for additional time consuming analytical characterization. We utilized this technique to analyze 243,000 µm3 of mouse liver tissue for the presence of PEG-R-Si-Au-NPs. Nanoparticles injected into the mice intravenously via the tail-vein accumulated in the liver while those injected intrarectally did not, indicating that they remain in the colon and do not pass through the colon wall into the systemic circulation. PMID:23803218

Systematic approach to study of thinly and thickly sectioned melanoma tissues with scanning acoustic microscopy

NASA Astrophysics Data System (ADS)

Miyasaka, C.; Tittmann, B. R.; Tutwiler, R.; Tian, Y.; Maeva, E.; Shum, D.

2010-03-01

The present study is to investigate the feasibility of applying in-vivo acoustic microscopy to the analysis of cancerous tissue. The study was implemented with mechanical scanning reflection acoustic microscope (SAM) by the following procedures. First, we ultrasonically visualized thick sections of normal and tumor tissues to determine the lowest transducer frequency required for cellular imaging. We used skin for normal tissue and the tumor was a malignant melanoma. Thin sections of the tissue were also studied with the optical and high-frequency-ultrasonic imaging for pathological evaluation. Secondly, we ultrasonically visualized subsurface cellular details of thin tissue specimens with different modes (i.e., pulse and tone-burst wave modes) to obtain the highest quality ultrasonic images. The objective is to select the best mode for the future design of a future SAM for in-vivo examination. Thirdly, we developed a mathematical modeling technique based on an angular spectrum approach for improving image processing and comparing numerical to experimental results.

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

NASA Astrophysics Data System (ADS)

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

1998-03-01

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

Effects of pipette modulation and imaging distances on ion currents measured with scanning ion conductance microscopy (SICM).

PubMed

Chen, Chiao-Chen; Baker, Lane A

2011-01-07

Local conductance variations can be estimated by measuring ion current magnitudes with scanning ion conductance microscopy (SICM). Factors which influence image quality and quantitation of ion currents measured with SICM have been evaluated. Specifically, effects of probe-sample separation and pipette modulation have been systematically studied for the case of imaging conductance variations at pores in a polymer membrane under transmembrane concentration gradients. The influence of probe-sample separation on ion current images was evaluated using distance-modulated (ac) feedback. Approach curves obtained using non-modulated (dc) feedback were also recorded to determine the relative influence of pipette-generated convection by comparison of ion currents measured with both ac and dc feedback modes. To better interpret results obtained, comparison to a model based on a disk-shaped geometry for nanopores in the membrane, as well as relevant position-dependent parameters of the experiment is described. These results advance our current understanding of conductance measurements with SICM.

Environmental Scanning Electron Microscope Imaging of Vesicle Systems.

PubMed

Perrie, Yvonne; Ali, Habib; Kirby, Daniel J; Mohammed, Afzal U R; McNeil, Sarah E; Vangala, Anil

2017-01-01

The structural characteristics of liposomes have been widely investigated and there is certainly a strong understanding of their morphological characteristics. Imaging of these systems, using techniques such as freeze-fracturing methods, transmission electron microscopy, and cryo-electron imaging, has allowed us to appreciate their bilayer structures and factors which can influence this. However, there are few methods which all us to study these systems in their natural hydrated state; commonly the liposomes are visualized after drying, staining, and/or fixation of the vesicles. Environmental Scanning Electron Microscopy (ESEM) offers the ability to image a liposome in its hydrated state without the need for prior sample preparation. Within our studies we were the first to use ESEM to study liposomes and niosomes and we have been able to dynamically follow the hydration of lipid films and changes in liposome suspensions as water condenses on to, or evaporates from, the sample in real time. This provides insight into the resistance of liposomes to coalescence during dehydration, thereby providing an alternative assay of liposome formulation and stability.

Theoretical characterisation of point defects on a MoS2 monolayer by scanning tunnelling microscopy.

PubMed

González, C; Biel, B; Dappe, Y J

2016-03-11

Different S and Mo vacancies as well as their corresponding antisite defects in a free-standing MoS2 monolayer are analysed by means of scanning tunnelling microscopy (STM) simulations. Our theoretical methodology, based on the Keldysh nonequilibrium Green function formalism within the density functional theory (DFT) approach, is applied to simulate STM images for different voltages and tip heights. Combining the geometrical and electronic effects, all features of the different STM images can be explained, providing a valuable guide for future experiments. Our results confirm previous reports on S atom imaging, but also reveal a strong dependence on the applied bias for vacancies and antisite defects that include extra S atoms. By contrast, when additional Mo atoms cover the S vacancies, the MoS2 gap vanishes and a bias-independent bright protrusion is obtained in the STM image. Finally, we show that the inclusion of these point defects promotes the emergence of reactive dangling bonds that may act as efficient adsorption sites for external adsorbates.

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

Buck, E.C.; Dietz, N.L.; Bates, J.K.

Uranium contaminated soils from the Fernald Operation Site, Ohio, have been examined by a combination of optical microscopy, scanning electron microscopy with backscattered electron detection (SEM/BSE), and analytical electron microscopy (AEM). A method is described for preparing of transmission electron microscopy (TEM) thin sections by ultramicrotomy. By using these thin sections, SEM and TEM images can be compared directly. Uranium was found in iron oxides, silicates (soddyite), phosphates (autunites), and fluorite. Little uranium was associated with clays. The distribution of uranium phases was found to be inhomogeneous at the microscopic level.

Status of the Nanoscopium scanning nanoprobe beamline of Synchrotron Soleil

NASA Astrophysics Data System (ADS)

Somogyi, A.; Medjoubi, K.; Kewish, C. M.; Leroux, V.; Ribbens, M.; Baranton, G.; Polack, F.; Samama, J. P.

2013-09-01

The Nanoscopium 155 m-long scanning nanoprobe beamline of Synchrotron Soleil (St Aubin, France) is dedicated to quantitative multi-modal imaging. Dedicated experimental stations, working in consecutive operation mode, will provide coherent scatter imaging and spectro-microscopy techniques in the 5-20 keV energy range for various user communities. Next to fast scanning, cryogenic cooling will reduce the radiation damage of sensitive samples during the measurements. Nanoscopium is in the construction phase, the first user experiments are expected in 2014. The main characteristics of the beamline and an overview of its status are given in this contribution.