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

PubMed

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

2017-11-01

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

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

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

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

Multimodality optical imaging of embryonic heart microstructure

PubMed Central

Yelin, Ronit; Yelin, Dvir; Oh, Wang-Yuhl; Yun, Seok H.; Boudoux, Caroline; Vakoc, Benjamin J.; Bouma, Brett E.; Tearney, Guillermo J.

2009-01-01

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 µm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass. PMID:18163837

Multimodality optical imaging of embryonic heart microstructure.

PubMed

Yelin, Ronit; Yelin, Dvir; Oh, Wang-Yuhl; Yun, Seok H; Boudoux, Caroline; Vakoc, Benjamin J; Bouma, Brett E; Tearney, Guillermo J

2007-01-01

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 microm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass.

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

PubMed

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

2017-01-23

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

Optical properties of mouse brain tissue after optical clearing with FocusClear™

NASA Astrophysics Data System (ADS)

Moy, Austin J.; Capulong, Bernard V.; Saager, Rolf B.; Wiersma, Matthew P.; Lo, Patrick C.; Durkin, Anthony J.; Choi, Bernard

2015-09-01

Fluorescence microscopy is commonly used to investigate disease progression in biological tissues. Biological tissues, however, are strongly scattering in the visible wavelengths, limiting the application of fluorescence microscopy to superficial (<200 μm) regions. Optical clearing, which involves incubation of the tissue in a chemical bath, reduces the optical scattering in tissue, resulting in increased tissue transparency and optical imaging depth. The goal of this study was to determine the time- and wavelength-resolved dynamics of the optical scattering properties of rodent brain after optical clearing with FocusClear™. Light transmittance and reflectance of 1-mm mouse brain sections were measured using an integrating sphere before and after optical clearing and the inverse adding doubling algorithm used to determine tissue optical scattering. The degree of optical clearing was quantified by calculating the optical clearing potential (OCP), and the effects of differing OCP were demonstrated using the optical histology method, which combines tissue optical clearing with optical imaging to visualize the microvasculature. We observed increased tissue transparency with longer optical clearing time and an analogous increase in OCP. Furthermore, OCP did not vary substantially between 400 and 1000 nm for increasing optical clearing durations, suggesting that optical histology can improve ex vivo visualization of several fluorescent probes.

Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy.

PubMed

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

2015-01-01

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

Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy

PubMed Central

Sternberg, Jenna R.; Wyart, Claire; Emiliani, Valentina

2015-01-01

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

Non-interferometric deep optical resolution photoacoustic remote sensing microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

HajiReza, Parsin H.; Bell, Kevan L.; Shi, Wei; Zemp, Roger J.

2017-03-01

A novel all-optical non-contact photoacoustic microscopy system is introduced. The confocal configuration is used to ensure detection of initial pressure shock wave-induced intensity reflections at the subsurface origin where pressures are largest. Phantom studies confirm signal dependence on optical absorption, index-contrast, and excitation fluence. Taking advantage of a focused1310 nm interrogation beam, the penetration depth of the system is improved to 2mm for an optical resolution system. High signal-to-noise ratios (>60dB) with 2.5 cm working distance from the objective lens to the sample is achieved. Real-time in-vivo imaging of microvasculature and melanoma tumors are demonstrated.

Al nanogrid electrode for ultraviolet detectors.

PubMed

Ding, G; Deng, J; Zhou, L; Gan, Q; Hwang, J C M; Dierolf, V; Bartoli, F J; Mazuir, C; Schoenfeld, W V

2011-09-15

Optical properties of Al nanogrids of different pitches and gaps were investigated both theoretically and experimentally. Three-dimensional finite-difference time-domain simulation predicted that surface plasmons at the air/Al interface would enhance ultraviolet transmission through the subwavelength gaps of the nanogrid, making it an effective electrode on GaN-based photodetectors to compensate for the lack of transparent electrode and high p-type doping. The predicted transmission enhancement was verified by confocal scanning optical microscopy performed at 365 nm. The quality of the nanogrids fabricated by electron-beam lithography was verified by near-field scanning optical microscopy and scanning electron microscopy. Based on the results, the pitch and gap of the nanogrids can be optimized for the best trade-off between electrical conductivity and optical transmission at different wavelengths. Based on different cutoff wavelengths, the nanogrids can also double as a filter to render photodetectors solar-blind.

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

PubMed

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

2013-09-01

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

Photon gating in four-dimensional ultrafast electron microscopy.

PubMed

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

2015-10-20

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

Photon gating in four-dimensional ultrafast electron microscopy

PubMed Central

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

2015-01-01

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

Visual-servoing optical microscopy

DOEpatents

Callahan, Daniel E.; Parvin, Bahram

2009-06-09

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

Visual-servoing optical microscopy

DOEpatents

Callahan, Daniel E [Martinez, CA; Parvin, Bahram [Mill Valley, CA

2011-05-24

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

Visual-servoing optical microscopy

DOEpatents

Callahan, Daniel E; Parvin, Bahram

2013-10-01

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

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

PubMed

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

2016-03-10

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

Axial Colocalization of Single Molecules with Nanometer Accuracy Using Metal-Induced Energy Transfer.

PubMed

Isbaner, Sebastian; Karedla, Narain; Kaminska, Izabela; Ruhlandt, Daja; Raab, Mario; Bohlen, Johann; Chizhik, Alexey; Gregor, Ingo; Tinnefeld, Philip; Enderlein, Jörg; Tsukanov, Roman

2018-04-11

Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups.

Optical approach to the salivary pellicle

NASA Astrophysics Data System (ADS)

Baek, Jae Ho; Krasieva, Tatiana; Tang, Shuo; Ahn, Yehchan; Kim, Chang Soo; Vu, Diana; Chen, Zhongping; Wilder-Smith, Petra

2009-07-01

The salivary pellicle plays an important role in oral physiology, yet noninvasive in situ characterization and mapping of this layer remains elusive. The goal of this study is to develop an optical approach for the real-time, noninvasive mapping and characterization of salivary pellicles using optical coherence tomography (OCT) and optical coherence microscopy (OCM). The long-term goals are to improve diagnostic capabilities in the oral cavity, gain a better understanding of physiological and pathological processes related to the oral hard tissues, and monitor treatment responses. A salivary pellicle is incubated on small enamel cubes using human whole saliva. OCT and OCM imaging occurs at 0, 10, 30, 60 min, and 24 h. For some imaging, spherical gold nanoparticles (15 nm) are added to determine whether this would increase the optical signal from the pellicle. Multiphoton microscopy (MPM) provides the baseline information. In the saliva-incubated samples, a surface signal from the developing pellicle is visible in OCT images. Pellicle ``islands'' form, which increase in complexity over time until they merge to form a continuous layer over the enamel surface. Noninvasive, in situ time-based pellicle formation on the enamel surface is visualized and characterized using optical imaging.

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

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

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.

Ultrafast time-resolved photoemission of a metallic tip/substrate junction

NASA Astrophysics Data System (ADS)

Meng, Xiang; Jin, Wencan; Yang, Hao; Dadap, Jerry; Osgood, Richard; Camillone, Nicholas, III

The strong near-field enhancement of metallic-tip nanostructures has attracted great interest in scanning microscopy techniques, such as surface-enhanced Raman scattering, near-field scanning optical microscopy and tip-enhanced nonlinear imaging. In this talk, we use a full vectorial 3D-FDTD method to investigate the spatial characteristics of the optical field confinement and localization between a tungsten nanoprobe and an infinite planar silver substrate, with two-color ultrafast laser excitation scheme. The degree of two-color excited field enhancement, geometry dependence, the exact mechanism of optical tip-substrate coupling and tip-substrate plasmon resonances are significant in understanding the electrodynamical responses at tip-substrate junction. The demonstrated measurements with subpicosecond time and subnanometer spatial resolution suggest a new approach to ultrafast time-resolved measurements of surface electron dynamics. DE-FG 02-90-ER-14104; DE-FG 02-04-ER-46157.

Fibre-optic nonlinear optical microscopy and endoscopy.

PubMed

Fu, L; Gu, M

2007-06-01

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

Label-free hyperspectral nonlinear optical microscopy of the biofuel micro-algae Haematococcus Pluvialis

PubMed Central

Barlow, Aaron M.; Slepkov, Aaron D.; Ridsdale, Andrew; McGinn, Patrick J.; Stolow, Albert

2014-01-01

We consider multi-modal four-wave mixing microscopies to be ideal tools for the in vivo study of carotenoid distributions within the important biofuel microalgae Haematococcus pluvialis. We show that hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy generates non-invasive, quantitative real-time concentrations maps of intracellular carotenoid distributions in live algae. PMID:25360358

Local optical control of ferromagnetism and chemical potential in a topological insulator

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

Yeats, Andrew L.; Mintun, Peter J.; Pan, Yu

Many proposed experiments involving topological insulators (TIs) require spatial control over time-reversal symmetry and chemical potential. We demonstrate reconfigurable micron-scale optical control of both magnetization (which breaks time-reversal symmetry) and chemical potential in ferromagnetic thin films of Cr-(Bi,Sb) 2Te 3 grown on SrTiO 3. By optically modulating the coercivity of the films, we write and erase arbitrary patterns in their remanent magnetization, which we then image with Kerr microscopy. Additionally, by optically manipulating a space charge layer in the underlying SrTiO 3 substrates, we control the local chemical potential of the films. This optical gating effect allows us to writemore » and erase p-n junctions in the films, which we study with photocurrent microscopy. Both effects are persistent and may be patterned and imaged independently on a few-micron scale. As a result, dynamic optical control over both magnetization and chemical potential of a TI may be useful in efforts to understand and control the edge states predicted at magnetic domain walls in quantum anomalous Hall insulators.« less

Local optical control of ferromagnetism and chemical potential in a topological insulator

DOE PAGES

Yeats, Andrew L.; Mintun, Peter J.; Pan, Yu; ...

2017-09-12

Many proposed experiments involving topological insulators (TIs) require spatial control over time-reversal symmetry and chemical potential. We demonstrate reconfigurable micron-scale optical control of both magnetization (which breaks time-reversal symmetry) and chemical potential in ferromagnetic thin films of Cr-(Bi,Sb) 2Te 3 grown on SrTiO 3. By optically modulating the coercivity of the films, we write and erase arbitrary patterns in their remanent magnetization, which we then image with Kerr microscopy. Additionally, by optically manipulating a space charge layer in the underlying SrTiO 3 substrates, we control the local chemical potential of the films. This optical gating effect allows us to writemore » and erase p-n junctions in the films, which we study with photocurrent microscopy. Both effects are persistent and may be patterned and imaged independently on a few-micron scale. As a result, dynamic optical control over both magnetization and chemical potential of a TI may be useful in efforts to understand and control the edge states predicted at magnetic domain walls in quantum anomalous Hall insulators.« less

PREFACE: Ultrafast biophotonics Ultrafast biophotonics

NASA Astrophysics Data System (ADS)

Gu, Min; Reid, Derryck; Ben-Yakar, Adela

2010-08-01

The use of light to explore biology can be traced to the first observations of tissue made with early microscopes in the mid-seventeenth century, and has today evolved into the discipline which we now know as biophotonics. This field encompasses a diverse range of activities, each of which shares the common theme of exploiting the interaction of light with biological material. With the rapid advancement of ultrafast optical technologies over the last few decades, ultrafast lasers have increasingly found applications in biophotonics, to the extent that the distinctive new field of ultrafast biophotonics has now emerged, where robust turnkey ultrafast laser systems are facilitating cutting-edge studies in the life sciences to take place in everyday laboratories. The broad spectral bandwidths, precision timing resolution, low coherence and high peak powers of ultrafast optical pulses provide unique opportunities for imaging and manipulating biological systems. Time-resolved studies of bio-molecular dynamics exploit the short pulse durations from such lasers, while other applications such as optical coherence tomography benefit from the broad optical bandwidths possible by using super-continuum generation and additionally allowing for high speed imaging with speeds as high as 47 000 scans per second. Continuing progress in laser-system technology is accelerating the adoption of ultrafast techniques across the life sciences, both in research laboratories and in clinical applications, such as laser-assisted in situ keratomileusis (LASIK) eye surgery. Revolutionizing the field of optical microscopy, two-photon excitation fluorescence (TPEF) microscopy has enabled higher spatial resolution with improved depth penetration into biological specimens. Advantages of this nonlinear optical process include: reduced photo-interactions, allowing for extensive imaging time periods; simultaneously exciting multiple fluorescent molecules with only one excitation wavelength; and reduced chromatic aberration effects. These extensive advantages have led to further exploration of nonlinear processes including second-harmonic generation (SHG) microscopy and third-harmonic generation (THG) microscopy. Second-harmonic generation has provided biologists with an extremely powerful tool for generating contrast in biological imaging, with the additional benefit of non-invasive three-dimensional imaging. The recent popularity of THG microscopy is largely due to the fact that three-dimensional imaging is achievable without the need for any labels, but rather relying on the intrinsic properties of the biological specimen itself. This optical nonlinear technique has attracted much attention recently from the biological community due to its non-invasive capabilities. Users of ultrafast lasers in the biological and medical fields are becoming a fast-growing community, employing pulse-shaping microscopy, resolution-enhancing microscopy techniques, linear and nonlinear micro-spectroscopy, functional deep-tissue imaging, optical coherence tomography, nonlinear fluorescence microscopy, molecular imaging and control, harmonic microscopy and femtosecond lifetime imaging, for cutting-edge research concerning the interaction of light with biological dynamics. The adaptability of ultrafast lasers to interact with a large array of materials through nonlinear excitation has enabled precise control of laser fluence allowing for highly localized material interactions, permitting micro-structured fabricated surfaces. The resultant multi-dimensional fabricated micro-structures are capable of replicating and/or manipulating microenvironments for controlled cell biology. In this special issue of Journal of Optics readers have a chance to view a collection of new contributions to the growing research field of ultrafast biophotonics. They are presented with recent advances in ultrafast technology applied to biological and medical investigations, where topics include advances in the visualization and identification of photo-reaction dynamics of biological functions under relevant physiological conditions, theoretically proposed imaging designs for obtaining super-resolved optical sectioned images in single exposures and fabricated micro-structured surfaces for biological micro-environments. We hope the collection will stimulate innovative new research in this growing field by showcasing new techniques for the visualization and manipulation of complex biological systems using linear and and nonlinear optical processes. Professor Min Gu would like to acknowledge Dr Betty Kouskousis for her contribution and support towards this editorial.

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.

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

NASA Astrophysics Data System (ADS)

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

2018-04-01

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

Optimizing Nanoscale Quantitative Optical Imaging of Subfield Scattering Targets

PubMed Central

Henn, Mark-Alexander; Barnes, Bryan M.; Zhou, Hui; Sohn, Martin; Silver, Richard M.

2016-01-01

The full 3-D scattered field above finite sets of features has been shown to contain a continuum of spatial frequency information, and with novel optical microscopy techniques and electromagnetic modeling, deep-subwavelength geometrical parameters can be determined. Similarly, by using simulations, scattering geometries and experimental conditions can be established to tailor scattered fields that yield lower parametric uncertainties while decreasing the number of measurements and the area of such finite sets of features. Such optimized conditions are reported through quantitative optical imaging in 193 nm scatterfield microscopy using feature sets up to four times smaller in area than state-of-the-art critical dimension targets. PMID:27805660

Measurement and modelization of silica opal optical properties

NASA Astrophysics Data System (ADS)

Avoine, Amaury; Hong, Phan Ngoc; Frederich, Hugo; Aregahegn, Kifle; Bénalloul, Paul; Coolen, Laurent; Schwob, Catherine; Thu Nga, Pham; Gallas, Bruno; Maître, Agnès

2014-03-01

We present the synthesis process and optical characterization of artificial silica opals. The specular reflection spectra are analyzed and compared to band structure calculations and finite difference time domain (FDTD) simulations. The silica optical index is a key parameter to correctly describe an opal and is usually not known and treated as a free parameter. Here we propose a method to infer the silica index, as well as the silica spheres diameter, from the reflection spectra and we validate it by comparison with two independent infrared methods for the index and, scanning electron microscopy (SEM) and atomic force microscopy (AFM) measurements for the spheres diameter.

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

PubMed

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

2016-03-01

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

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

PubMed

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

2016-06-20

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

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

PubMed Central

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

2014-01-01

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

Electromagnetic-Optical Coherence Tomography Guidance of Transbronchial Solitary Pulmonary Nodule Biopsy

DTIC Science & Technology

2016-04-01

6 1. INTRODUCTION Lung cancer is the leading cause of cancer related death accounting for more deaths than breast , prostate and colon...the cancer has spread, at which time patients have little chance of cure. Macroscopic imaging modalities including CT and bronchoscopy have made...Electromagnetic Navigation , Biopsy Guidance, Optical Microscopy, Optical Coherence Tomography, Lung Cancer , Optical needle. 3. OVERALL PROJECT SUMMARY

Super-resolution optical telescopes with local light diffraction shrinkage

PubMed Central

Wang, Changtao; Tang, Dongliang; Wang, Yanqin; Zhao, Zeyu; Wang, Jiong; Pu, Mingbo; Zhang, Yudong; Yan, Wei; Gao, Ping; Luo, Xiangang

2015-01-01

Suffering from giant size of objective lenses and infeasible manipulations of distant targets, telescopes could not seek helps from present super-resolution imaging, such as scanning near-field optical microscopy, perfect lens and stimulated emission depletion microscopy. In this paper, local light diffraction shrinkage associated with optical super-oscillatory phenomenon is proposed for real-time and optically restoring super-resolution imaging information in a telescope system. It is found that fine target features concealed in diffraction-limited optical images of a telescope could be observed in a small local field of view, benefiting from a relayed metasurface-based super-oscillatory imaging optics in which some local Fourier components beyond the cut-off frequency of telescope could be restored. As experimental examples, a minimal resolution to 0.55 of Rayleigh criterion is obtained, and imaging complex targets and large targets by superimposing multiple local fields of views are demonstrated as well. This investigation provides an access for real-time, incoherent and super-resolution telescopes without the manipulation of distant targets. More importantly, it gives counterintuitive evidence to the common knowledge that relayed optics could not deliver more imaging details than objective systems. PMID:26677820

Doppler optical coherence microscopy and tomography applied to inner ear mechanics

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

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

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

Review of advanced imaging techniques

PubMed Central

Chen, Yu; Liang, Chia-Pin; Liu, Yang; Fischer, Andrew H.; Parwani, Anil V.; Pantanowitz, Liron

2012-01-01

Pathology informatics encompasses digital imaging and related applications. Several specialized microscopy techniques have emerged which permit the acquisition of digital images (“optical biopsies”) at high resolution. Coupled with fiber-optic and micro-optic components, some of these imaging techniques (e.g., optical coherence tomography) are now integrated with a wide range of imaging devices such as endoscopes, laparoscopes, catheters, and needles that enable imaging inside the body. These advanced imaging modalities have exciting diagnostic potential and introduce new opportunities in pathology. Therefore, it is important that pathology informaticists understand these advanced imaging techniques and the impact they have on pathology. This paper reviews several recently developed microscopic techniques, including diffraction-limited methods (e.g., confocal microscopy, 2-photon microscopy, 4Pi microscopy, and spatially modulated illumination microscopy) and subdiffraction techniques (e.g., photoactivated localization microscopy, stochastic optical reconstruction microscopy, and stimulated emission depletion microscopy). This article serves as a primer for pathology informaticists, highlighting the fundamentals and applications of advanced optical imaging techniques. PMID:22754737

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

PubMed

Gu, Min; Fu, Ling

2006-02-06

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

Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning

PubMed Central

Silva, Susana F.; Domingues, José Paulo

2018-01-01

Time-gated fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to assess the biochemistry of cells and tissues. When applied to living thick samples, it is hampered by the lack of optical sectioning and the need of acquiring many images for an accurate measurement of fluorescence lifetimes. Here, we report on the use of processing techniques to overcome these limitations, minimizing the acquisition time, while providing optical sectioning. We evaluated the application of the HiLo and the rapid lifetime determination (RLD) techniques for accurate measurement of fluorescence lifetimes with optical sectioning. HiLo provides optical sectioning by combining the high-frequency content from a standard image, obtained with uniform illumination, with the low-frequency content of a second image, acquired using structured illumination. Our results show that HiLo produces optical sectioning on thick samples without degrading the accuracy of the measured lifetimes. We also show that instrument response function (IRF) deconvolution can be applied with the RLD technique on HiLo images, improving greatly the accuracy of the measured lifetimes. These results open the possibility of using the RLD technique with pulsed diode laser sources to determine accurately fluorescence lifetimes in the subnanosecond range on thick multilayer samples, providing that offline processing is allowed. PMID:29599938

Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning.

PubMed

Silva, Susana F; Domingues, José Paulo; Morgado, António Miguel

2018-01-01

Time-gated fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to assess the biochemistry of cells and tissues. When applied to living thick samples, it is hampered by the lack of optical sectioning and the need of acquiring many images for an accurate measurement of fluorescence lifetimes. Here, we report on the use of processing techniques to overcome these limitations, minimizing the acquisition time, while providing optical sectioning. We evaluated the application of the HiLo and the rapid lifetime determination (RLD) techniques for accurate measurement of fluorescence lifetimes with optical sectioning. HiLo provides optical sectioning by combining the high-frequency content from a standard image, obtained with uniform illumination, with the low-frequency content of a second image, acquired using structured illumination. Our results show that HiLo produces optical sectioning on thick samples without degrading the accuracy of the measured lifetimes. We also show that instrument response function (IRF) deconvolution can be applied with the RLD technique on HiLo images, improving greatly the accuracy of the measured lifetimes. These results open the possibility of using the RLD technique with pulsed diode laser sources to determine accurately fluorescence lifetimes in the subnanosecond range on thick multilayer samples, providing that offline processing is allowed.

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

NASA Astrophysics Data System (ADS)

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

2017-03-01

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

Multilayer mounting for long-term light sheet microscopy of zebrafish.

PubMed

Weber, Michael; Mickoleit, Michaela; Huisken, Jan

2014-02-27

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

PubMed Central

Weber, Michael; Mickoleit, Michaela; Huisken, Jan

2014-01-01

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology. PMID:24637614

The 2015 super-resolution microscopy roadmap

NASA Astrophysics Data System (ADS)

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

2015-11-01

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

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

NASA Astrophysics Data System (ADS)

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

2007-09-01

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

Spatially and temporally resolved exciton dynamics and transport in single nanostructures and assemblies

NASA Astrophysics Data System (ADS)

Huang, Libai

2015-03-01

The frontier in solar energy conversion now lies in learning how to integrate functional entities across multiple length scales to create optimal devices. To address this new frontier, I will discuss our recent efforts on elucidating multi-scale energy transfer, migration, and dissipation processes with simultaneous femtosecond temporal resolution and nanometer spatial resolution. We have developed ultrafast microscopy that combines ultrafast spectroscopy with optical microscopy to map exciton dynamics and transport with simultaneous ultrafast time resolution and diffraction-limited spatial resolution. We have employed pump-probe transient absorption microscopy to elucidate morphology and structure dependent exciton dynamics and transport in single nanostructures and molecular assemblies. More specifically, (1) We have applied transient absorption microscopy (TAM) to probe environmental and structure dependent exciton relaxation pathways in sing-walled carbon nanotubes (SWNTs) by mapping dynamics in individual pristine SWNTs with known structures. (2) We have systematically measured and modeled the optical properties of the Frenkel excitons in self-assembled porphyrin tubular aggregates that represent an analog to natural photosynthetic antennae. Using a combination of ultrafast optical microscopy and stochastic exciton modeling, we address exciton transport and relaxation pathways, especially those related to disorder.

Optical Coherence Microscopy

NASA Astrophysics Data System (ADS)

Aguirre, Aaron D.; Zhou, Chao; Lee, Hsiang-Chieh; Ahsen, Osman O.; Fujimoto, James G.

Cellular imaging of human tissues remains an important advance for many clinical applications of optical coherence tomography (OCT). Imaging cells with traditional OCT systems has not been possible due to the limited transverse resolution of such techniques. Optical coherence microscopy (OCM) refers to OCT methods that achieve high transverse resolution to visualize cells and subcellular features. This chapter provides a comprehensive discussion of the rationale for cellular imaging in human tissues as well as a review of the key technological advances required to achieve it. Time domain and Fourier domain OCM approaches are described with an emphasis on state of the art system designs, including miniaturized endoscopic imaging probes. Clinical applications are discussed and multiple examples of cellular imaging in human tissues are provided.

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

PubMed

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

2013-09-09

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

Real-time optically sectioned wide-field microscopy employing structured light illumination and a CMOS detector

NASA Astrophysics Data System (ADS)

Mitic, Jelena; Anhut, Tiemo; Serov, Alexandre; Lasser, Theo; Bourquin, Stephane

2003-07-01

Real-time optically sectioned microscopy is demonstrated using an AC-sensitive detection concept realized with smart CMOS image sensor and structured light illumination by a continuously moving periodic pattern. We describe two different detection systems based on CMOS image sensors for the detection and on-chip processing of the sectioned images in real time. A region-of-interest is sampled at high frame rate. The demodulated signal delivered by the detector corresponds to the depth discriminated image of the sample. The measured FWHM of the axial response depends on the spatial frequency of the projected grid illumination and is in the μm-range. The effect of using broadband incoherent illumination is discussed. The performance of these systems is demonstrated by imaging technical as well as biological samples.

Simulated microsurgery monitoring using intraoperative multimodal surgical microscopy

NASA Astrophysics Data System (ADS)

Lee, Donghyun; Lee, Changho; Kim, Sehui; Zhou, Qifa; Kim, Jeehyun; Kim, Chulhong

2016-03-01

We have developed an intraoperative multimodal surgical microscopy system that provides simultaneous real-time enlarged surface views and subsurface anatomic information during surgeries by integrating spectral domain optical coherence tomography (SD-OCT), optical-resolution photoacoustic microscopy (OR-PAM), and conventional surgical microscopy. By sharing the same optical path, both OCT and PAM images were simultaneously acquired. Additionally, the custom-made needle-type transducer received the generated PA signals enabling convenient surgical operation without using a water bath. Using a simple augmented device, the OCT and PAM images were projected on the view plane of the surgical microscope. To quantify the performance of our system, we measured spatial resolutions of our system. Then, three microsurgery simulation and analysis were processed: (1) ex vivo needle tracking and monitoring injection of carbon particles in biological tissues, (2) in vivo needle tracking and monitoring injection of carbon particles in tumor-bearing mice, and (3) in vivo guiding of melanoma removal in melanoma-bearing mice. The results indicate that this triple modal system is useful for intraoperative purposes, and can potentially be a vital tool in microsurgeries.

Scanning near-field optical microscopy.

PubMed

Vobornik, Dusan; Vobornik, Slavenka

2008-02-01

An average human eye can see details down to 0,07 mm in size. The ability to see smaller details of the matter is correlated with the development of the science and the comprehension of the nature. Today's science needs eyes for the nano-world. Examples are easily found in biology and medical sciences. There is a great need to determine shape, size, chemical composition, molecular structure and dynamic properties of nano-structures. To do this, microscopes with high spatial, spectral and temporal resolution are required. Scanning Near-field Optical Microscopy (SNOM) is a new step in the evolution of microscopy. The conventional, lens-based microscopes have their resolution limited by diffraction. SNOM is not subject to this limitation and can offer up to 70 times better resolution.

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.

Dual-dimensional microscopy: real-time in vivo three-dimensional observation method using high-resolution light-field microscopy and light-field display.

PubMed

Kim, Jonghyun; Moon, Seokil; Jeong, Youngmo; Jang, Changwon; Kim, Youngmin; Lee, Byoungho

2018-06-01

Here, we present dual-dimensional microscopy that captures both two-dimensional (2-D) and light-field images of an in-vivo sample simultaneously, synthesizes an upsampled light-field image in real time, and visualizes it with a computational light-field display system in real time. Compared with conventional light-field microscopy, the additional 2-D image greatly enhances the lateral resolution at the native object plane up to the diffraction limit and compensates for the image degradation at the native object plane. The whole process from capturing to displaying is done in real time with the parallel computation algorithm, which enables the observation of the sample's three-dimensional (3-D) movement and direct interaction with the in-vivo sample. We demonstrate a real-time 3-D interactive experiment with Caenorhabditis elegans. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

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.

Image fidelity improvement in digital holographic microscopy using optical phase conjugation

NASA Astrophysics Data System (ADS)

Chan, Huang-Tian; Chew, Yang-Kun; Shiu, Min-Tzung; Chang, Chi-Ching

2018-01-01

With respect to digital holography, techniques in suppressing noises derived from reference arm are maturely developed. However, techniques for the object counterpart are not being well developed. Optical phase conjugation technique was believed to be a promising method for this interest. A 0°-cut BaTiO3 photorefractive crystal was involved in self-pumped phase conjugation scheme, and was employed to in-line digital holographic microscopy, in both transmission-type and reflection-type configuration. On pure physical compensation basis, results revealed that the image fidelity was improved substantially with 2.9096 times decrease in noise level and 3.5486 times increase in the ability to discriminate noise on average, by suppressing the scattering noise prior to recording stage.

Nonlinear Optical Spectroscopy of Two-Dimensional Materials

NASA Astrophysics Data System (ADS)

Cui, Qiannan

Nonlinear optical properties of two-dimensional (2D) materials, such as transition metal dichalcogenides (TMDs), graphene, black phosphorus, and so on, play a key role of understanding nanoscale light-matter interactions, as well as developing nanophotonics applications from solar cells to quantum computation. With ultrafast lasers, we experimentally study nonlinear optical properties of 2D materials. Employing transient absorption microscopy, we study several members of 2D materials, such as WSe2, TiS3 and ReS2. The dynamical saturable absorption process of 2D excitons is spatiotemporally resolved. Intrinsic parameters of these 2D materials, such as exciton lifetime, exciton diffusion coefficient, and exciton mobility, are effectively measured. Especially, in-plane anisotropy of transient absorption and diffusive transport is observed for 2D excitons in monolayer ReS2, demonstrating the in-plane degree of freedom. Furthermore, with quantum interference and control nanoscopy, we all-optically inject, detect and manipulate nanoscale ballistic charge currents in a ReS2 thin film. By tuning the phase difference between one photon absorption and two photon absorption transition paths, sub-picosecond timescale of ballistic currents is coherently controlled for the first time in TMDs. In addition, the spatial resolution is two-order of magnitude smaller than optical diffraction limit. The second-order optical nonlinearity of 2D monolayers is resolved by second harmonic generation (SHG) microscopy. We measure the second-order susceptibility of monolayer MoS 2. The angular dependence of SHG in monolayer MoS2 shows strong symmetry dependence on its crystal lattice structure. Hence, second harmonic generation microscopy can serve as a powerful tool to noninvasively determine the crystalline directions of 2D monolayers. The real and imaginary parts of third-order optical nonlinearity of 2D monolayers are resolved by third harmonic generation (THG) microscopy and two-photon transient absorption microscopy, respectively. With third harmonic generation microscopy, we observe strong and anisotropic THG in monolayer and multilayer ReS2. Comparing with 2D materials with hexagonal lattice, such as MoS2, the third-order susceptibility is higher by one order of magnitude in ReS2 with a distorted 1T structure. The in-plane anisotropy of THG is attributed to the lattice distortion in ReS2 after comparing with a symmetry analysis. With two-photon transient absorption microscopy, we observe a giant two-photon absorption coefficient of monolayer WS2.

Ultrafast optical pulse delivery with fibers for nonlinear microscopy

PubMed Central

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

2008-01-01

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

Optics clustered to output unique solutions: A multi-laser facility for combined single molecule and ensemble microscopy

NASA Astrophysics Data System (ADS)

Clarke, David T.; Botchway, Stanley W.; Coles, Benjamin C.; Needham, Sarah R.; Roberts, Selene K.; Rolfe, Daniel J.; Tynan, Christopher J.; Ward, Andrew D.; Webb, Stephen E. D.; Yadav, Rahul; Zanetti-Domingues, Laura; Martin-Fernandez, Marisa L.

2011-09-01

Optics clustered to output unique solutions (OCTOPUS) is a microscopy platform that combines single molecule and ensemble imaging methodologies. A novel aspect of OCTOPUS is its laser excitation system, which consists of a central core of interlocked continuous wave and pulsed laser sources, launched into optical fibres and linked via laser combiners. Fibres are plugged into wall-mounted patch panels that reach microscopy end-stations in adjacent rooms. This allows multiple tailor-made combinations of laser colours and time characteristics to be shared by different end-stations minimising the need for laser duplications. This setup brings significant benefits in terms of cost effectiveness, ease of operation, and user safety. The modular nature of OCTOPUS also facilitates the addition of new techniques as required, allowing the use of existing lasers in new microscopes while retaining the ability to run the established parts of the facility. To date, techniques interlinked are multi-photon/multicolour confocal fluorescence lifetime imaging for several modalities of fluorescence resonance energy transfer (FRET) and time-resolved anisotropy, total internal reflection fluorescence, single molecule imaging of single pair FRET, single molecule fluorescence polarisation, particle tracking, and optical tweezers. Here, we use a well-studied system, the epidermal growth factor receptor network, to illustrate how OCTOPUS can aid in the investigation of complex biological phenomena.

Visible light optical coherence microscopy imaging of the mouse cortex with femtoliter volume resolution

NASA Astrophysics Data System (ADS)

Merkle, Conrad W.; Chong, Shau Poh; Kho, Aaron M.; Zhu, Jun; Kholiqov, Oybek; Dubra, Alfredo; Srinivasan, Vivek J.

2018-02-01

Most flying-spot Optical Coherence Tomography (OCT) and Optical Coherence Microscopy (OCM) systems use a symmetric confocal geometry, where the detection path retraces the illumination path starting from and ending with the spatial mode of a single mode optical fiber. Here, we describe a visible light OCM instrument that breaks this symmetry to improve transverse resolution without sacrificing collection efficiency in scattering tissue. This was achieved by overfilling a 0.3 numerical aperture (NA) water immersion objective on the illumination path, while maintaining a conventional Gaussian mode detection path (1/e2 intensity diameter 0.82 Airy disks), enabling 1.1 μm full-width at half-maximum (FWHM) transverse resolution. At the same time, a 0.9 μm FWHM axial resolution in tissue, achieved by a broadband visible light source, enabled femtoliter volume resolution. We characterized this instrument according to paraxial coherent microscopy theory, and then used it to image the meningeal layers, intravascular red blood cell-free layer, and myelinated axons in the mouse neocortex in vivo through the thinned skull. Finally, by introducing a 0.8 NA water immersion objective, we improved the lateral resolution to 0.44 μm FWHM, which provided a volumetric resolution of 0.2 fL, revealing cell bodies in cortical layer I of the mouse brain with OCM for the first time.

[Cornea imagery and keratitis caused by processionary caterpillar hairs].

PubMed

Fournier, I; Saleh, M; Beynat, J; Creuzot-Garcher, C; Bourcier, T; Speeg-Schatz, C

2011-03-01

With their ability to migrate into the cornea and release toxins, caterpillar hairs can induce different clinical presentations such as conjunctivitis, keratoconjunctivitis, uveitis, and less frequently vitreoretinal inflammation (hyalitis, papillitis, macular edema). We report a case that occurred in Alsace (France) in a 13-years-old boy presenting with keratitis caused by caterpillar hairs. We localized them in the cornea, for the first time, using confocal microscopy and anterior segment spectral optical coherence tomography. Confocal microscopy and spectral optical coherence tomography can be useful for diagnosis and follow-up of this disease. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Study of optically trapped living Trypanosoma cruzi/Trypanosoma rangeli - Rhodnius prolixus interactions by real time confocal images using CdSe quantum dots

NASA Astrophysics Data System (ADS)

de Thomaz, A. A.; Almeida, D. B.; Faustino, W. M.; Jacob, G. J.; Fontes, A.; Barbosa, L. C.; Cesar, C. L.; Stahl, C. V.; Santos-Mallet, J. R.; Gomes, S. A. O.; Feder, D.

2008-08-01

One of the fundamental goals in biology is to understand the interplay between biomolecules of different cells. This happen, for example, in the first moments of the infection of a vector by a parasite that results in the adherence to the cell walls. To observe this kind of event we used an integrated Optical Tweezers and Confocal Microscopy tool. This tool allow us to use the Optical Tweezers to trigger the adhesion of the Trypanosoma cruzi and Trypanosoma rangeli parasite to the intestine wall cells and salivary gland of the Rhodnius prolixus vector and to, subsequently observe the sequence of events by confocal fluorescence microscopy under optical forces stresses. We kept the microorganism and vector cells alive using CdSe quantum dot staining. Besides the fact that Quantum Dots are bright vital fluorescent markers, the absence of photobleaching allow us to follow the events in time for an extended period. By zooming to the region of interested we have been able to acquire confocal images at the 2 to 3 frames per second rate.

SCANNING NEAR-FIELD OPTICAL MICROSCOPY

PubMed Central

Vobornik, Dušan; Vobornik, Slavenka

2008-01-01

An average human eye can see details down to 0,07 mm in size. The ability to see smaller details of the matter is correlated with the development of the science and the comprehension of the nature. Today’s science needs eyes for the nano-world. Examples are easily found in biology and medical sciences. There is a great need to determine shape, size, chemical composition, molecular structure and dynamic properties of nano-structures. To do this, microscopes with high spatial, spectral and temporal resolution are required. Scanning Near-field Optical Microscopy (SNOM) is a new step in the evolution of microscopy. The conventional, lens-based microscopes have their resolution limited by diffraction. SNOM is not subject to this limitation and can offer up to 70 times better resolution. PMID:18318675

Developing single-laser sources for multimodal coherent anti-Stokes Raman scattering microscopy

NASA Astrophysics Data System (ADS)

Pegoraro, Adrian Frank

Coherent anti-Stokes Raman scattering (CARS) microscopy has developed rapidly and is opening the door to new types of experiments. This work describes the development of new laser sources for CARS microscopy and their use for different applications. It is specifically focused on multimodal nonlinear optical microscopy—the simultaneous combination of different imaging techniques. This allows us to address a diverse range of applications, such as the study of biomaterials, fluid inclusions, atherosclerosis, hepatitis C infection in cells, and ice formation in cells. For these applications new laser sources are developed that allow for practical multimodal imaging. For example, it is shown that using a single Ti:sapphire oscillator with a photonic crystal fiber, it is possible to develop a versatile multimodal imaging system using optimally chirped laser pulses. This system can perform simultaneous two photon excited fluorescence, second harmonic generation, and CARS microscopy. The versatility of the system is further demonstrated by showing that it is possible to probe different Raman modes using CARS microscopy simply by changing a time delay between the excitation beams. Using optimally chirped pulses also enables further simplification of the laser system required by using a single fiber laser combined with nonlinear optical fibers to perform effective multimodal imaging. While these sources are useful for practical multimodal imaging, it is believed that for further improvements in CARS microscopy sensitivity, new excitation schemes are necessary. This has led to the design of a new, high power, extended cavity oscillator that should be capable of implementing new excitation schemes for CARS microscopy as well as other techniques. Our interest in multimodal imaging has led us to other areas of research as well. For example, a fiber-coupling scheme for signal collection in the forward direction is demonstrated that allows for fluorescence lifetime imaging without significant temporal distortion. Also highlighted is an imaging artifact that is unique to CARS microscopy that can alter image interpretation, especially when using multimodal imaging. By combining expertise in nonlinear optics, laser development, fiber optics, and microscopy, we have developed systems and techniques that will be of benefit for multimodal CARS microscopy.

Ultrafast time-stretch imaging at 932 nm through a new highly-dispersive fiber

PubMed Central

Wei, Xiaoming; Kong, Cihang; Sy, Samuel; Ko, Ho; Tsia, Kevin K.; Wong, Kenneth K. Y.

2016-01-01

Optical glass fiber has played a key role in the development of modern optical communication and attracted the biotechnology researcher’s great attention because of its properties, such as the wide bandwidth, low attenuation and superior flexibility. For ultrafast optical imaging, particularly, it has been utilized to perform MHz time-stretch imaging with diffraction-limited resolutions, which is also known as serial time-encoded amplified microscopy (STEAM). Unfortunately, time-stretch imaging with dispersive fibers has so far mostly been demonstrated at the optical communication window of 1.5 μm due to lack of efficient dispersive optical fibers operating at the shorter wavelengths, particularly at the bio-favorable window, i.e., <1.0 μm. Through fiber-optic engineering, here we demonstrate a 7.6-MHz dual-color time-stretch optical imaging at bio-favorable wavelengths of 932 nm and 466 nm. The sensitivity at such a high speed is experimentally identified in a slow data-streaming manner. To the best of our knowledge, this is the first time that all-optical time-stretch imaging at ultrahigh speed, high sensitivity and high chirping rate (>1 ns/nm) has been demonstrated at a bio-favorable wavelength window through fiber-optic engineering. PMID:28018737

Ultrafast time-stretch imaging at 932 nm through a new highly-dispersive fiber.

PubMed

Wei, Xiaoming; Kong, Cihang; Sy, Samuel; Ko, Ho; Tsia, Kevin K; Wong, Kenneth K Y

2016-12-01

Optical glass fiber has played a key role in the development of modern optical communication and attracted the biotechnology researcher's great attention because of its properties, such as the wide bandwidth, low attenuation and superior flexibility. For ultrafast optical imaging, particularly, it has been utilized to perform MHz time-stretch imaging with diffraction-limited resolutions, which is also known as serial time-encoded amplified microscopy (STEAM). Unfortunately, time-stretch imaging with dispersive fibers has so far mostly been demonstrated at the optical communication window of 1.5 μm due to lack of efficient dispersive optical fibers operating at the shorter wavelengths, particularly at the bio-favorable window, i.e., <1.0 μm. Through fiber-optic engineering, here we demonstrate a 7.6-MHz dual-color time-stretch optical imaging at bio-favorable wavelengths of 932 nm and 466 nm. The sensitivity at such a high speed is experimentally identified in a slow data-streaming manner. To the best of our knowledge, this is the first time that all-optical time-stretch imaging at ultrahigh speed, high sensitivity and high chirping rate (>1 ns/nm) has been demonstrated at a bio-favorable wavelength window through fiber-optic engineering.

Real-Time Nanoscopy by Using Blinking Enhanced Quantum Dots

PubMed Central

Watanabe, Tomonobu M.; Fukui, Shingo; Jin, Takashi; Fujii, Fumihiko; Yanagida, Toshio

2010-01-01

Superresolution optical microscopy (nanoscopy) is of current interest in many biological fields. Superresolution optical fluctuation imaging, which utilizes higher-order cumulant of fluorescence temporal fluctuations, is an excellent method for nanoscopy, as it requires neither complicated optics nor illuminations. However, it does need an impractical number of images for real-time observation. Here, we achieved real-time nanoscopy by modifying superresolution optical fluctuation imaging and enhancing the fluctuation of quantum dots. Our developed quantum dots have higher blinking than commercially available ones. The fluctuation of the blinking improved the resolution when using a variance calculation for each pixel instead of a cumulant calculation. This enabled us to obtain microscopic images with 90-nm and 80-ms spatial-temporal resolution by using a conventional fluorescence microscope without any optics or devices. PMID:20923631

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

NASA Astrophysics Data System (ADS)

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

2006-03-01

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

Numerical Simulation of Partially-Coherent Broadband Optical Imaging Using the FDTD Method

PubMed Central

Çapoğlu, İlker R.; White, Craig A.; Rogers, Jeremy D.; Subramanian, Hariharan; Taflove, Allen; Backman, Vadim

2012-01-01

Rigorous numerical modeling of optical systems has attracted interest in diverse research areas ranging from biophotonics to photolithography. We report the full-vector electromagnetic numerical simulation of a broadband optical imaging system with partially-coherent and unpolarized illumination. The scattering of light from the sample is calculated using the finite-difference time-domain (FDTD) numerical method. Geometrical optics principles are applied to the scattered light to obtain the intensity distribution at the image plane. Multilayered object spaces are also supported by our algorithm. For the first time, numerical FDTD calculations are directly compared to and shown to agree well with broadband experimental microscopy results. PMID:21540939

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

PubMed Central

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

2010-01-01

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

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

Characterization of passive polymer optical waveguides

NASA Astrophysics Data System (ADS)

Joehnck, Matthias; Kalveram, Stefan; Lehmacher, Stefan; Pompe, Guido; Rudolph, Stefan; Neyer, Andreas; Hofstraat, Johannes W.

1999-05-01

The characterization of monomode passive polymer optical devices fabricated according to the POPCORN technology by methods originated from electron, ion and optical spectroscopy is summarized. Impacts of observed waveguide perturbations on the optical characteristics of the waveguide are evaluated. In the POPCORN approach optical components for telecommunication applications are fabricated by photo-curing of liquid halogenated (meth)acrylates which have been applied on moulded thermoplastic substrates. For tuning of waveguide material refractive indices with respect to the substrate refractive index frequently comonomer mixtures are used. The polymerization characteristics, especially the polymerization kinetics of individual monomers, determine the formation of copolymers. Therefore the unsaturation as function of UV-illumination time in the formation of halogenated homo- and copolymers has been examined. From different suitable copolymer system, after characterization of their glass transition temperatures, their curing behavior and their refractive indices as function of the monomer ratios, monomode waveguides applying PMMA substrates have been fabricated. To examine the materials composition also in the 6 X 6 micrometers 2 waveguides they have been visualized by transmission electron microscopy. With this method e.g. segregation phenomena could be observed in the waveguide cross section characterization as well. The optical losses in monomode waveguides caused by segregation and other materials induce defects like micro bubbles formed as a result of shrinkage have been quantized by return loss measurements. Defects causing scattering could be observed by convocal laser scanning microscopy and by conventional light microscopy.

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

PubMed

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

2015-09-01

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

Electric-field induced surface instabilities of soft dielectrics and their effects on optical transmittance and scattering

NASA Astrophysics Data System (ADS)

Shian, Samuel; Kjeer, Peter; Clarke, David R.

2018-03-01

When a voltage is applied to a percolative, mechanically compliant mat of carbon nanotubes (CNTs) on a smooth elastomer bilayer attached to an ITO coated glass substrate, the in-line optical transmittance decreases with increasing voltage. Two regimes of behavior have been identified based on optical scattering, bright field optical microscopy, and confocal optical microscopy. In the low field regime, the electric field produces a spatially inhomogeneous surface deformation of the elastomer that causes local variations in optical refraction and modulates the light transmittance. The spatial variation is associated with the distribution of the CNTs over the surface. At higher fields, above a threshold voltage, an array of pits in the surface form by a nucleation and growth mechanism and these also scatter light. The formation of pits, and creases, in the thickness of the elastomer, is due to a previously identified electro-mechanical surface instability. When the applied voltage is decreased from its maximum, the transmittance returns to its original value although there is a transmittance hysteresis and a complicated time response. When the applied voltage exceeds the threshold voltage, there can be remnant optical contrast associated with creasing of the elastomer and the recovery time appears to be dependent on local jamming of CNTs in areas where the pits formed. A potential application of this work as an electrically tunable privacy window or camouflaging devices is demonstrated.

Massively parallel data processing for quantitative total flow imaging with optical coherence microscopy and tomography

NASA Astrophysics Data System (ADS)

Sylwestrzak, Marcin; Szlag, Daniel; Marchand, Paul J.; Kumar, Ashwin S.; Lasser, Theo

2017-08-01

We present an application of massively parallel processing of quantitative flow measurements data acquired using spectral optical coherence microscopy (SOCM). The need for massive signal processing of these particular datasets has been a major hurdle for many applications based on SOCM. In view of this difficulty, we implemented and adapted quantitative total flow estimation algorithms on graphics processing units (GPU) and achieved a 150 fold reduction in processing time when compared to a former CPU implementation. As SOCM constitutes the microscopy counterpart to spectral optical coherence tomography (SOCT), the developed processing procedure can be applied to both imaging modalities. We present the developed DLL library integrated in MATLAB (with an example) and have included the source code for adaptations and future improvements. Catalogue identifier: AFBT_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AFBT_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GNU GPLv3 No. of lines in distributed program, including test data, etc.: 913552 No. of bytes in distributed program, including test data, etc.: 270876249 Distribution format: tar.gz Programming language: CUDA/C, MATLAB. Computer: Intel x64 CPU, GPU supporting CUDA technology. Operating system: 64-bit Windows 7 Professional. Has the code been vectorized or parallelized?: Yes, CPU code has been vectorized in MATLAB, CUDA code has been parallelized. RAM: Dependent on users parameters, typically between several gigabytes and several tens of gigabytes Classification: 6.5, 18. Nature of problem: Speed up of data processing in optical coherence microscopy Solution method: Utilization of GPU for massively parallel data processing Additional comments: Compiled DLL library with source code and documentation, example of utilization (MATLAB script with raw data) Running time: 1,8 s for one B-scan (150 × faster in comparison to the CPU data processing time)

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

PubMed Central

Weigert, Martin; Bundschuh, Sebastian T.

2018-01-01

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

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

PubMed Central

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

2012-01-01

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

Improved localization accuracy in stochastic super-resolution fluorescence microscopy by K-factor image deshadowing

PubMed Central

Ilovitsh, Tali; Meiri, Amihai; Ebeling, Carl G.; Menon, Rajesh; Gerton, Jordan M.; Jorgensen, Erik M.; Zalevsky, Zeev

2013-01-01

Localization of a single fluorescent particle with sub-diffraction-limit accuracy is a key merit in localization microscopy. Existing methods such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) achieve localization accuracies of single emitters that can reach an order of magnitude lower than the conventional resolving capabilities of optical microscopy. However, these techniques require a sparse distribution of simultaneously activated fluorophores in the field of view, resulting in larger time needed for the construction of the full image. In this paper we present the use of a nonlinear image decomposition algorithm termed K-factor, which reduces an image into a nonlinear set of contrast-ordered decompositions whose joint product reassembles the original image. The K-factor technique, when implemented on raw data prior to localization, can improve the localization accuracy of standard existing methods, and also enable the localization of overlapping particles, allowing the use of increased fluorophore activation density, and thereby increased data collection speed. Numerical simulations of fluorescence data with random probe positions, and especially at high densities of activated fluorophores, demonstrate an improvement of up to 85% in the localization precision compared to single fitting techniques. Implementing the proposed concept on experimental data of cellular structures yielded a 37% improvement in resolution for the same super-resolution image acquisition time, and a decrease of 42% in the collection time of super-resolution data with the same resolution. PMID:24466491

Dual Optical Levers for Atomic Force Microscopy

NASA Astrophysics Data System (ADS)

Kawakatsu, Hideki; Bleuler, Hannes; Saito, Takashi; Hiroshi, Kougami

1995-06-01

Development of micro machined cantilever and optical lever detection system has greatly facilitated the operation of atomic force microscopy. However, since the detection system measures only the deflection of the cantilever at one set point where the laser beam is focused, care must be taken in implementing force control or in interpreting the acquired data. In this paper, a dual optical lever detection system is introduced, which has the potential to resolve the deformation of the cantilever with multidegree of freedom and thus detect the position of the tip end point with resolution in the 10 pm order. The detection system proved to be effective in real-time monitoring of the behavior of the tip end point while scanning, and in explaining the scanning direction dependence of the acquired images.

Combining near-field scanning optical microscopy with spectral interferometry for local characterization of the optical electric field in photonic structures.

PubMed

Trägårdh, Johanna; Gersen, Henkjan

2013-07-15

We show how a combination of near-field scanning optical microscopy with crossed beam spectral interferometry allows a local measurement of the spectral phase and amplitude of light propagating in photonic structures. The method only requires measurement at the single point of interest and at a reference point, to correct for the relative phase of the interferometer branches, to retrieve the dispersion properties of the sample. Furthermore, since the measurement is performed in the spectral domain, the spectral phase and amplitude could be retrieved from a single camera frame, here in 70 ms for a signal power of less than 100 pW limited by the dynamic range of the 8-bit camera. The method is substantially faster than most previous time-resolved NSOM methods that are based on time-domain interferometry, which also reduced problems with drift. We demonstrate how the method can be used to measure the refractive index and group velocity in a waveguide structure.

Rotary-scanning optical resolution photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Qi, Weizhi; Xi, Lei

2016-10-01

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

High-frame-rate imaging of biological samples with optoacoustic micro-tomography

NASA Astrophysics Data System (ADS)

Deán-Ben, X. Luís.; López-Schier, Hernán.; Razansky, Daniel

2018-02-01

Optical microscopy remains a major workhorse in biological discovery despite the fact that light scattering limits its applicability to depths of ˜ 1 mm in scattering tissues. Optoacoustic imaging has been shown to overcome this barrier by resolving optical absorption with microscopic resolution in significantly deeper regions. Yet, the time domain is paramount for the observation of biological dynamics in living systems that exhibit fast motion. Commonly, acquisition of microscopy data involves raster scanning across the imaged volume, which significantly limits temporal resolution in 3D. To overcome these limitations, we have devised a fast optoacoustic micro-tomography (OMT) approach based on simultaneous acquisition of 3D image data with a high-density hemispherical ultrasound array having effective detection bandwidth around 25 MHz. We performed experiments by imaging tissue-mimicking phantoms and zebrafish larvae, demonstrating that OMT can provide nearly cellular resolution and imaging speed of 100 volumetric frames per second. As opposed to other optical microscopy techniques, OMT is a hybrid method that resolves optical absorption contrast acoustically using unfocused light excitation. Thus, no penetration barriers are imposed by light scattering in deep tissues, suggesting it as a powerful approach for multi-scale functional and molecular imaging applications.

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.

Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging

NASA Astrophysics Data System (ADS)

Errico, Claudia; Pierre, Juliette; Pezet, Sophie; Desailly, Yann; Lenkei, Zsolt; Couture, Olivier; Tanter, Mickael

2015-11-01

Non-invasive imaging deep into organs at microscopic scales remains an open quest in biomedical imaging. Although optical microscopy is still limited to surface imaging owing to optical wave diffusion and fast decorrelation in tissue, revolutionary approaches such as fluorescence photo-activated localization microscopy led to a striking increase in resolution by more than an order of magnitude in the last decade. In contrast with optics, ultrasonic waves propagate deep into organs without losing their coherence and are much less affected by in vivo decorrelation processes. However, their resolution is impeded by the fundamental limits of diffraction, which impose a long-standing trade-off between resolution and penetration. This limits clinical and preclinical ultrasound imaging to a sub-millimetre scale. Here we demonstrate in vivo that ultrasound imaging at ultrafast frame rates (more than 500 frames per second) provides an analogue to optical localization microscopy by capturing the transient signal decorrelation of contrast agents—inert gas microbubbles. Ultrafast ultrasound localization microscopy allowed both non-invasive sub-wavelength structural imaging and haemodynamic quantification of rodent cerebral microvessels (less than ten micrometres in diameter) more than ten millimetres below the tissue surface, leading to transcranial whole-brain imaging within short acquisition times (tens of seconds). After intravenous injection, single echoes from individual microbubbles were detected through ultrafast imaging. Their localization, not limited by diffraction, was accumulated over 75,000 images, yielding 1,000,000 events per coronal plane and statistically independent pixels of ten micrometres in size. Precise temporal tracking of microbubble positions allowed us to extract accurately in-plane velocities of the blood flow with a large dynamic range (from one millimetre per second to several centimetres per second). These results pave the way for deep non-invasive microscopy in animals and humans using ultrasound. We anticipate that ultrafast ultrasound localization microscopy may become an invaluable tool for the fundamental understanding and diagnostics of various disease processes that modify the microvascular blood flow, such as cancer, stroke and arteriosclerosis.

Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging.

PubMed

Errico, Claudia; Pierre, Juliette; Pezet, Sophie; Desailly, Yann; Lenkei, Zsolt; Couture, Olivier; Tanter, Mickael

2015-11-26

Non-invasive imaging deep into organs at microscopic scales remains an open quest in biomedical imaging. Although optical microscopy is still limited to surface imaging owing to optical wave diffusion and fast decorrelation in tissue, revolutionary approaches such as fluorescence photo-activated localization microscopy led to a striking increase in resolution by more than an order of magnitude in the last decade. In contrast with optics, ultrasonic waves propagate deep into organs without losing their coherence and are much less affected by in vivo decorrelation processes. However, their resolution is impeded by the fundamental limits of diffraction, which impose a long-standing trade-off between resolution and penetration. This limits clinical and preclinical ultrasound imaging to a sub-millimetre scale. Here we demonstrate in vivo that ultrasound imaging at ultrafast frame rates (more than 500 frames per second) provides an analogue to optical localization microscopy by capturing the transient signal decorrelation of contrast agents--inert gas microbubbles. Ultrafast ultrasound localization microscopy allowed both non-invasive sub-wavelength structural imaging and haemodynamic quantification of rodent cerebral microvessels (less than ten micrometres in diameter) more than ten millimetres below the tissue surface, leading to transcranial whole-brain imaging within short acquisition times (tens of seconds). After intravenous injection, single echoes from individual microbubbles were detected through ultrafast imaging. Their localization, not limited by diffraction, was accumulated over 75,000 images, yielding 1,000,000 events per coronal plane and statistically independent pixels of ten micrometres in size. Precise temporal tracking of microbubble positions allowed us to extract accurately in-plane velocities of the blood flow with a large dynamic range (from one millimetre per second to several centimetres per second). These results pave the way for deep non-invasive microscopy in animals and humans using ultrasound. We anticipate that ultrafast ultrasound localization microscopy may become an invaluable tool for the fundamental understanding and diagnostics of various disease processes that modify the microvascular blood flow, such as cancer, stroke and arteriosclerosis.

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

NASA Astrophysics Data System (ADS)

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

2018-05-01

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

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

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

Ramanathan, Nathan Muruganathan; Darling, Seth B.

2015-01-01

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

Stochastic Optical Reconstruction Microscopy (STORM).

PubMed

Xu, Jianquan; Ma, Hongqiang; Liu, Yang

2017-07-05

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

Superresolution upgrade for confocal spinning disk systems using image scanning microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Isbaner, Sebastian; Hähnel, Dirk; Gregor, Ingo; Enderlein, Jörg

2017-02-01

Confocal Spinning Disk Systems are widely used for 3D cell imaging because they offer the advantage of optical sectioning at high framerates and are easy to use. However, as in confocal microscopy, the imaging resolution is diffraction limited, which can be theoretically improved by a factor of 2 using the principle of Image Scanning Microscopy (ISM) [1]. ISM with a Confocal Spinning Disk setup (CSDISM) has been shown to improve contrast as well as lateral resolution (FWHM) from 201 +/- 20 nm to 130 +/- 10 nm at 488 nm excitation. A minimum total acquisition time of one second per ISM image makes this method highly suitable for 3D live cell imaging [2]. Here, we present a multicolor implementation of CSDISM for the popular Micro-Manager Open Source Microscopy platform. Since changes in the optical path are not necessary, this will allow any researcher to easily upgrade their standard Confocal Spinning Disk system at remarkable low cost ( 5000 USD) with an ISM superresolution option. [1]. Müller, C.B. and Enderlein, J. Image Scanning Microscopy. Physical Review Letters 104, (2010). [2]. Schulz, O. et al. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy. Proceedings of the National Academy of Sciences of the United States of America 110, 21000-5 (2013).

Optofluidic time-stretch quantitative phase microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Wu, Yi; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Lee, Sangwook; Isozaki, Akihiro; Li, Ming; Jiang, Yiyue; Yasumoto, Atsushi; Di Carlo, Dino; Tanaka, Yo; Yatomi, Yutaka; Ozeki, Yasuyuki; Goda, Keisuke

2018-03-01

Innovations in optical microscopy have opened new windows onto scientific research, industrial quality control, and medical practice over the last few decades. One of such innovations is optofluidic time-stretch quantitative phase microscopy - an emerging method for high-throughput quantitative phase imaging that builds on the interference between temporally stretched signal and reference pulses by using dispersive properties of light in both spatial and temporal domains in an interferometric configuration on a microfluidic platform. It achieves the continuous acquisition of both intensity and phase images with a high throughput of more than 10,000 particles or cells per second by overcoming speed limitations that exist in conventional quantitative phase imaging methods. Applications enabled by such capabilities are versatile and include characterization of cancer cells and microalgal cultures. In this paper, we review the principles and applications of optofluidic time-stretch quantitative phase microscopy and discuss its future perspective. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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

2018-05-28

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

Confocal Raman microscopy for monitoring chemical reactions on single optically trapped, solid-phase support particles.

PubMed

Houlne, Michael P; Sjostrom, Christopher M; Uibel, Rory H; Kleimeyer, James A; Harris, Joel M

2002-09-01

Optical trapping of small structures is a powerful tool for the manipulation and investigation of colloidal and particulate materials. The tight focus excitation requirements of optical trapping are well suited to confocal Raman microscopy. In this work, an inverted confocal Raman microscope is developed for studies of chemical reactions on single, optically trapped particles and applied to reactions used in solid-phase peptide synthesis. Optical trapping and levitation allow a particle to be moved away from the coverslip and into solution, avoiding fluorescence interference from the coverslip. More importantly, diffusion of reagents into the particle is not inhibited by a surface, so that reaction conditions mimic those of particles dispersed in solution. Optical trapping and levitation also maintain optical alignment, since the particle is centered laterally along the optical axis and within the focal plane of the objective, where both optical forces and light collection are maximized. Hour-long observations of chemical reactions on individual, trapped silica particles are reported. Using two-dimensional least-squares analysis methods, the Raman spectra collected during the course of a reaction can be resolved into component contributions. The resolved spectra of the time-varying species can be observed, as they bind to or cleave from the particle surface.

Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline

PubMed Central

Madden, Jeremy T.; Toth, Scott J.; Dettmar, Christopher M.; Newman, Justin A.; Oglesbee, Robert A.; Hedderich, Hartmut G.; Everly, R. Michael; Becker, Michael; Ronau, Judith A.; Buchanan, Susan K.; Cherezov, Vadim; Morrow, Marie E.; Xu, Shenglan; Ferguson, Dale; Makarov, Oleg; Das, Chittaranjan; Fischetti, Robert; Simpson, Garth J.

2013-01-01

Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼103–104-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase from Chromobacterium violaceum cPAH, Trichinella spiralis deubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied. PMID:23765294

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

PubMed

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

2012-01-01

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

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

NASA Astrophysics Data System (ADS)

Upputuri, Paul Kumar; Pramanik, Manojit

2017-12-01

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

A robotized six degree of freedom stage for optical microscopy

NASA Astrophysics Data System (ADS)

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

2013-04-01

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

Decalin-assisted light emitting porous Si formation and its optical, surface and morphological properties

NASA Astrophysics Data System (ADS)

Karatutlu, Ali; Istengir, Sumeyra; Cosgun, Sedat; Seker, Isa; Unal, Bayram

2017-11-01

In this research paper, light emitting porous silicon (Lep-Si) samples were fabricated by a surfactant-mediated chemical stain etching solution in order to form homogenous luminescent nanostructures at room temperature. As an industrially important solvent, decalin (decahydronaphtalene) was used as a surfactant in the HF/HNO3 solutions in order to control the etching process. Morphological, surface and optical properties of the Lep-Si samples were examined using atomic force microscopy, X-ray photoelectron spectroscopy, photoluminescence (PL) spectroscopy, and laser scanning confocal microscopy (LSCM) techniques. These characterization techniques were correlated with the various etching times including depth dependent luminescence profiles for the first time. We report the optimum conditions for production of the most efficient Lep-Si using decalin (decahydronaphtalene) and possible structural origins of light emission using the depth dependent luminescence measurements.

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.

Real-time and non-invasive measurements of cell mechanical behaviour with optical coherence phase microscopy

NASA Astrophysics Data System (ADS)

Gillies, D.; Gamal, W.; Downes, A.; Reinwald, Y.; Yang, Y.; El Haj, A.; Bagnaninchi, P. O.

2017-02-01

There is an unmet need in tissue engineering for non-invasive, label-free monitoring of cell mechanical behaviour in their physiological environment. Here, we describe a novel optical coherence phase microscopy (OCPM) set-up which can map relative cell mechanical behaviour in monolayers and 3D systems non-invasively, and in real-time. 3T3 and MCF-7 cells were investigated, with MCF-7 demonstrating an increased response to hydrostatic stimulus indicating MCF-7 being softer than 3T3. Thus, OCPM shows the ability to provide qualitative data on cell mechanical behaviour. Quantitative measurements of 6% agarose beads have been taken with commercial Cell Scale Microsquisher system demonstrating that their mechanical properties are in the same order of magnitude of cells, indicating that this is an appropriate test sample for the novel method described.

Characterization of the Polycaprolactone Melt Crystallization: Complementary Optical Microscopy, DSC, and AFM Studies

PubMed Central

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

2014-01-01

The first stages of the crystallization of polycaprolactone (PCL) were studied using several techniques. The crystallization exotherms measured by differential scanning calorimetry (DSC) were analyzed and compared with results obtained by polarized optical microscopy (POM), rheology, and atomic force microscope (AFM). The experimental results suggest a strong influence of the observation scale. In particular, the AFM, even if limited on time scale, appears to be the most sensitive technique to detect the first stages of crystallization. On the contrary, at least in the case analysed in this work, rheology appears to be the least sensitive technique. DSC and POM provide closer results. This suggests that the definition of induction time in the polymer crystallization is a vague concept that, in any case, requires the definition of the technique used for its characterization. PMID:24523644

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

PubMed

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

2014-01-01

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

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

PubMed

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

2012-09-01

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

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.

Real-time digital signal processing in multiphoton and time-resolved microscopy

NASA Astrophysics Data System (ADS)

Wilson, Jesse W.; Warren, Warren S.; Fischer, Martin C.

2016-03-01

The use of multiphoton interactions in biological tissue for imaging contrast requires highly sensitive optical measurements. These often involve signal processing and filtering steps between the photodetector and the data acquisition device, such as photon counting and lock-in amplification. These steps can be implemented as real-time digital signal processing (DSP) elements on field-programmable gate array (FPGA) devices, an approach that affords much greater flexibility than commercial photon counting or lock-in devices. We will present progress toward developing two new FPGA-based DSP devices for multiphoton and time-resolved microscopy applications. The first is a high-speed multiharmonic lock-in amplifier for transient absorption microscopy, which is being developed for real-time analysis of the intensity-dependence of melanin, with applications in vivo and ex vivo (noninvasive histopathology of melanoma and pigmented lesions). The second device is a kHz lock-in amplifier running on a low cost (50-200) development platform. It is our hope that these FPGA-based DSP devices will enable new, high-speed, low-cost applications in multiphoton and time-resolved microscopy.

Diabetes screening by telecentric digital holographic microscopy.

PubMed

Doblas, A; Roche, E; Ampudia-Blasco, F J; Martínez-Corral, M; Saavedra, G; Garcia-Sucerquia, J

2016-03-01

Diabetes is currently the world's fastest growing chronic disease and it is caused by deficient production of insulin by the endocrine pancreas or by abnormal insulin action in peripheral tissues. This results in persistent hyperglycaemia that over time may produce chronic diabetic complications. Determination of glycated haemoglobin level is currently the gold standard method to evaluate and control sustained hyperglycaemia in diabetic people. This measurement is currently made by high-performance liquid chromatography, which is a complex chemical process that requires the extraction of blood from the antecubital vein. To reduce the complexity of that measurement, we propose a fully-optical technique that is based in the fact that there are changes in the optical properties of erythrocytes due to the presence of glucose-derived adducts in the haemoglobin molecule. To evaluate these changes, we propose to perform quantitative phase maps of erythrocytes by using telecentric digital holographic microscopy. Our experiments show that telecentric digital holographic microscopy allows detecting, almost in real time and from a single drop of blood, significant differences between erythrocytes of diabetic patients and healthy patients. Besides, our phase measurements are well correlated with the values of glycated haemoglobin and the blood glucose values. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Progress in the Correlative Atomic Force Microscopy and Optical Microscopy

PubMed Central

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

2017-01-01

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

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.

Attomicroscopy: from femtosecond to attosecond electron microscopy

NASA Astrophysics Data System (ADS)

Hassan, Mohammed Th

2018-02-01

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

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

PubMed

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

2013-01-01

Super-resolution three-dimensional (3D) optical microscopy has incomparable advantages over other high-resolution microscopic technologies, such as electron microscopy and atomic force microscopy, in the study of biological molecules, pathways and events in live cells and tissues. We present a novel approach of structured illumination microscopy (SIM) by using a digital micromirror device (DMD) for fringe projection and a low-coherence LED light for illumination. The lateral resolution of 90 nm and the optical sectioning depth of 120 μm were achieved. The maximum acquisition speed for 3D imaging in the optical sectioning mode was 1.6×10(7) pixels/second, which was mainly limited by the sensitivity and speed of the CCD camera. In contrast to other SIM techniques, the DMD-based LED-illumination SIM is cost-effective, ease of multi-wavelength switchable and speckle-noise-free. The 2D super-resolution and 3D optical sectioning modalities can be easily switched and applied to either fluorescent or non-fluorescent specimens.

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

PubMed Central

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

2013-01-01

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

Adaptive optical fluorescence microscopy.

PubMed

Ji, Na

2017-03-31

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

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

Madden, Jeremy T.; Toth, Scott J.; Dettmar, Christopher M.

Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ~10 3–10 4-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering andmore » analysis of phenylalanine hydroxylase fromChromobacterium violaceumcPAH,Trichinella spiralisdeubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied.« less

Solution-Processable transparent conducting electrodes via the self-assembly of silver nanowires for organic photovoltaic devices.

PubMed

Tugba Camic, B; Jeong Shin, Hee; Hasan Aslan, M; Basarir, Fevzihan; Choi, Hyosung

2018-02-15

Solution-processed transparent conducting electrodes (TCEs) were fabricated via the self-assembly deposition of silver nanowires (Ag NWs). Glass substrates modified with (3-aminopropyl)triethoxysilane (APTES) and (3-mercaptopropyl)trimethoxysilane (MPTES) were coated with Ag NWs for various deposition times, leading to three different Ag NWs samples (APTES-Ag NWs (PVP), MPTES-Ag NWs (PVP), and APTES-Ag NWs (COOH)). Controlling the deposition time produced Ag NWs monolayer thin films with different optical transmittance and sheet resistance. Post-annealing treatment improved their electrical conductivity. The Ag NWs films were successfully characterized using UV-Vis spectroscopy, field emission scanning electron microscopy, optical microscopy and four-point probe. Three Ag NWs films exhibited low sheet resistance of 4-19Ω/sq and high optical transmittance of 65-81% (at 550nm), which are comparable to those of commercial ITO electrode. We fabricated an organic photovoltaic device by using Ag NWs as the anode instead of ITO electrode, and optimized device with Ag NWs exhibited power conversion efficiency of 1.72%. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2012-01-01

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

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

PubMed Central

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

2014-01-01

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

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

New Technique for Fabrication of Scanning Single-Electron Transistor Microscopy Tips

NASA Astrophysics Data System (ADS)

Goodwin, Eric; Tessmer, Stuart

Fabrication of glass tips for Scanning Single-Electron Transistor Microscopy (SSETM) can be expensive, time consuming, and inconsistent. Various techniques have been tried, with varying levels of success in regards to cost and reproducibility. The main requirement for SSETM tips is to have a sharp tip ending in a micron-scale flat face to allow for deposition of a quantum dot. Drawing inspiration from methods used to create tips from optical fibers for Near-Field Scanning Optical Microscopes, our group has come up with a quick and cost effective process for creating SSETM tips. By utilizing hydrofluoric acid to etch the tips and oleic acid to guide the etch profile, optical fiber tips with appropriate shaping can be rapidly prepared. Once etched, electric leads are thermally evaporated onto each side of the tip, while an aluminum quantum dot is evaporated onto the face. Preliminary results using various metals, oxide layers, and lead thicknesses have proven promising.

Precise Spatially Selective Photothermolysis Using Modulated Femtosecond Lasers and Real-time Multimodal Microscopy Monitoring.

PubMed

Huang, Yimei; Lui, Harvey; Zhao, Jianhua; Wu, Zhenguo; Zeng, Haishan

2017-01-01

The successful application of lasers in the treatment of skin diseases and cosmetic surgery is largely based on the principle of conventional selective photothermolysis which relies strongly on the difference in the absorption between the therapeutic target and its surroundings. However, when the differentiation in absorption is not sufficient, collateral damage would occur due to indiscriminate and nonspecific tissue heating. To deal with such cases, we introduce a novel spatially selective photothermolysis method based on multiphoton absorption in which the radiant energy of a tightly focused near-infrared femtosecond laser beam can be directed spatially by aiming the laser focal point to the target of interest. We construct a multimodal optical microscope to perform and monitor the spatially selective photothermolysis. We demonstrate that precise alteration of the targeted tissue is achieved while leaving surrounding tissue intact by choosing appropriate femtosecond laser exposure with multimodal optical microscopy monitoring in real time.

Precise Spatially Selective Photothermolysis Using Modulated Femtosecond Lasers and Real-time Multimodal Microscopy Monitoring

PubMed Central

Huang, Yimei; Lui, Harvey; Zhao, Jianhua; Wu, Zhenguo; Zeng, Haishan

2017-01-01

The successful application of lasers in the treatment of skin diseases and cosmetic surgery is largely based on the principle of conventional selective photothermolysis which relies strongly on the difference in the absorption between the therapeutic target and its surroundings. However, when the differentiation in absorption is not sufficient, collateral damage would occur due to indiscriminate and nonspecific tissue heating. To deal with such cases, we introduce a novel spatially selective photothermolysis method based on multiphoton absorption in which the radiant energy of a tightly focused near-infrared femtosecond laser beam can be directed spatially by aiming the laser focal point to the target of interest. We construct a multimodal optical microscope to perform and monitor the spatially selective photothermolysis. We demonstrate that precise alteration of the targeted tissue is achieved while leaving surrounding tissue intact by choosing appropriate femtosecond laser exposure with multimodal optical microscopy monitoring in real time. PMID:28255346

Effect of silver thickness on structural, optical and morphological properties of nanocrystalline Ag/NiO thin films

NASA Astrophysics Data System (ADS)

Jalili, S.; Hajakbari, F.; Hojabri, A.

2018-03-01

Silver (Ag) nanolayers were deposited on nickel oxide (NiO) thin films by DC magnetron sputtering. The thickness of Ag layers was in range of 20-80 nm by variation of deposition time between 10 and 40 s. X-ray diffraction results showed that the crystalline properties of the Ag/NiO films improved by increasing the Ag film thickness. Also, atomic force microscopy and field emission scanning electron microscopy images demonstrated that the surface morphology of the films was highly affected by film thickness. The film thickness and the size of particles change by elevating the Ag deposition times. The composition of films was determined by Rutherford back scattering spectroscopy. The transmission of light was gradually reduced by augmentation of Ag films thickness. Furthermore; the optical band gap of the films was also calculated from the transmittance spectra.

Sub-10 fs Time-Resolved Vibronic Optical Microscopy

PubMed Central

2016-01-01

We introduce femtosecond wide-field transient absorption microscopy combining sub-10 fs pump and probe pulses covering the complete visible (500–650 nm) and near-infrared (650–950 nm) spectrum with diffraction-limited optical resolution. We demonstrate the capabilities of our system by reporting the spatially- and spectrally-resolved transient electronic response of MAPbI3–xClx perovskite films and reveal significant quenching of the transient bleach signal at grain boundaries. The unprecedented temporal resolution enables us to directly observe the formation of band-gap renormalization, completed in 25 fs after photoexcitation. In addition, we acquire hyperspectral Raman maps of TIPS pentacene films with sub-400 nm spatial and sub-15 cm–1 spectral resolution covering the 100–2000 cm–1 window. Our approach opens up the possibility of studying ultrafast dynamics on nanometer length and femtosecond time scales in a variety of two-dimensional and nanoscopic systems. PMID:27934055

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.

In Situ Identification of Nanoparticle Structural Information Using Optical Microscopy.

PubMed

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

2018-05-11

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

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

PubMed

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

2016-03-15

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

Communication: atomic force detection of single-molecule nonlinear optical vibrational spectroscopy.

PubMed

Saurabh, Prasoon; Mukamel, Shaul

2014-04-28

Atomic Force Microscopy (AFM) allows for a highly sensitive detection of spectroscopic signals. This has been first demonstrated for NMR of a single molecule and recently extended to stimulated Raman in the optical regime. We theoretically investigate the use of optical forces to detect time and frequency domain nonlinear optical signals. We show that, with proper phase matching, the AFM-detected signals closely resemble coherent heterodyne-detected signals. Applications are made to AFM-detected and heterodyne-detected vibrational resonances in Coherent Anti-Stokes Raman Spectroscopy (χ((3))) and sum or difference frequency generation (χ((2))).

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

NASA Astrophysics Data System (ADS)

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

2017-03-01

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

Optical gating and streaking of free electrons with sub-optical cycle precision

PubMed Central

Kozák, M.; McNeur, J.; Leedle, K. J.; Deng, H.; Schönenberger, N.; Ruehl, A.; Hartl, I.; Harris, J. S.; Byer, R. L.; Hommelhoff, P.

2017-01-01

The temporal resolution of ultrafast electron diffraction and microscopy experiments is currently limited by the available experimental techniques for the generation and characterization of electron bunches with single femtosecond or attosecond durations. Here, we present proof of principle experiments of an optical gating concept for free electrons via direct time-domain visualization of the sub-optical cycle energy and transverse momentum structure imprinted on the electron beam. We demonstrate a temporal resolution of 1.2±0.3 fs. The scheme is based on the synchronous interaction between electrons and the near-field mode of a dielectric nano-grating excited by a femtosecond laser pulse with an optical period duration of 6.5 fs. The sub-optical cycle resolution demonstrated here is promising for use in laser-driven streak cameras for attosecond temporal characterization of bunched particle beams as well as time-resolved experiments with free-electron beams. PMID:28120930

Confocal Raman microscopy supported by optical clearing treatment of the skin—influence on collagen hydration

NASA Astrophysics Data System (ADS)

Sdobnov, Anton Yu; Tuchin, Valery V.; Lademann, Juergen; E Darvin, Maxim

2017-07-01

Confocal Raman microscopy (CRM) is employed to study the skin physiology, drug permeation and skin disease monitoring. In order to increase the depth of investigations, the effect of optical clearing was observed on porcine ear skin ex vivo. The optical clearing agents (OCAs) glycerol and iohexol (Omnipaque™) were applied to the porcine ear skin and investigated by CRM after 30 and 60 min of treatment. The extent of optical clearing by utilizing concentrations of 70% glycerol and 100% Omnipaque™ was evaluated. The intensity of the skin-related Raman peaks significantly increased starting from the depth 160 µm for Omnipaque™ and 40 µm for glycerol (p  ⩽  0.05) after 60 min of treatment. The OCAs’ influence on the collagen hydration in the deep-located dermis was investigated. Both OCAs induce skin dehydration, but the effect of glycerol treatment (30 min and 60 min) is stronger. The obtained results demonstrate that with increasing the treatment time, both glycerol and Omnipaque™ solutions improve the optical clearing of porcine skin making the deep-located dermal regions able for investigations. At the used concentrations and time intervals, glycerol is more effective than Omnipaque™. However, Omnipaque™ is more promising than glycerol for future in vivo applications as it is an already approved pharmaceutic substance without any known impact on the skin structure.

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

PubMed

Scarpettini, A F; Bragas, A V

2015-01-01

Field-enhanced scanning optical microscopy relies on the design and fabrication of plasmonic probes which had to provide optical and chemical contrast at the nanoscale. In order to do so, the scattering containing the near-field information recorded in a field-enhanced scanning optical microscopy experiment, has to surpass the background light, always present due to multiple interferences between the macroscopic probe and sample. In this work, we show that when the probe-sample distance is modulated with very low amplitude, the higher the harmonic demodulation is, the better the ratio between the near-field signal and the interferometric background results. The choice of working at a given n harmonic is dictated by the experiment when the signal at the n + 1 harmonic goes below the experimental noise. We demonstrate that the optical contrast comes from the nth derivative of the near-field scattering, amplified by the interferometric background. By modelling the far and near field we calculate the probe-sample approach curves, which fit very well the experimental ones. After taking a great amount of experimental data for different probes and samples, we conclude with a table of the minimum enhancement factors needed to have optical contrast with field-enhanced scanning optical microscopy. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

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

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

Visualization of carrier dynamics in p(n)-type GaAs by scanning ultrafast electron microscopy

PubMed Central

Cho, Jongweon; Hwang, Taek Yong; Zewail, Ahmed H.

2014-01-01

Four-dimensional scanning ultrafast electron microscopy is used to investigate doping- and carrier-concentration-dependent ultrafast carrier dynamics of the in situ cleaved single-crystalline GaAs(110) substrates. We observed marked changes in the measured time-resolved secondary electrons depending on the induced alterations in the electronic structure. The enhancement of secondary electrons at positive times, when the electron pulse follows the optical pulse, is primarily due to an energy gain involving the photoexcited charge carriers that are transiently populated in the conduction band and further promoted by the electron pulse, consistent with a band structure that is dependent on chemical doping and carrier concentration. When electrons undergo sufficient energy loss on their journey to the surface, dark contrast becomes dominant in the image. At negative times, however, when the electron pulse precedes the optical pulse (electron impact), the dynamical behavior of carriers manifests itself in a dark contrast which indicates the suppression of secondary electrons upon the arrival of the optical pulse. In this case, the loss of energy of material’s electrons is by collisions with the excited carriers. These results for carrier dynamics in GaAs(110) suggest strong carrier–carrier scatterings which are mirrored in the energy of material’s secondary electrons during their migration to the surface. The approach presented here provides a fundamental understanding of materials probed by four-dimensional scanning ultrafast electron microscopy, and offers possibilities for use of this imaging technique in the study of ultrafast charge carrier dynamics in heterogeneously patterned micro- and nanostructured material surfaces and interfaces. PMID:24469803

Visualization of carrier dynamics in p(n)-type GaAs by scanning ultrafast electron microscopy.

PubMed

Cho, Jongweon; Hwang, Taek Yong; Zewail, Ahmed H

2014-02-11

Four-dimensional scanning ultrafast electron microscopy is used to investigate doping- and carrier-concentration-dependent ultrafast carrier dynamics of the in situ cleaved single-crystalline GaAs(110) substrates. We observed marked changes in the measured time-resolved secondary electrons depending on the induced alterations in the electronic structure. The enhancement of secondary electrons at positive times, when the electron pulse follows the optical pulse, is primarily due to an energy gain involving the photoexcited charge carriers that are transiently populated in the conduction band and further promoted by the electron pulse, consistent with a band structure that is dependent on chemical doping and carrier concentration. When electrons undergo sufficient energy loss on their journey to the surface, dark contrast becomes dominant in the image. At negative times, however, when the electron pulse precedes the optical pulse (electron impact), the dynamical behavior of carriers manifests itself in a dark contrast which indicates the suppression of secondary electrons upon the arrival of the optical pulse. In this case, the loss of energy of material's electrons is by collisions with the excited carriers. These results for carrier dynamics in GaAs(110) suggest strong carrier-carrier scatterings which are mirrored in the energy of material's secondary electrons during their migration to the surface. The approach presented here provides a fundamental understanding of materials probed by four-dimensional scanning ultrafast electron microscopy, and offers possibilities for use of this imaging technique in the study of ultrafast charge carrier dynamics in heterogeneously patterned micro- and nanostructured material surfaces and interfaces.

Photoacoustic spectral characterization of perfluorocarbon droplets

NASA Astrophysics Data System (ADS)

Strohm, Eric; Gorelikov, Ivan; Matsuura, Naomi; Kolios, Michael

2012-02-01

Perfluorocarbon droplets containing optical absorbing nanoparticles have been developed for use as theranostic agents (for both imaging and therapy) and as dual-mode contrast agents. Droplets can be used as photoacoustic contrast agents, vaporized via optical irradiation, then the resulting bubbles can be used as ultrasound imaging and therapeutic agents. The photoacoustic signals from micron-sized droplets containing silica coated gold nanospheres were measured using ultra-high frequencies (100-1000 MHz). The spectra of droplets embedded in a gelatin phantom were compared to a theoretical model which calculates the pressure wave from a spherical homogenous liquid undergoing thermoelastic expansion resulting from laser absorption. The location of the spectral features of the theoretical model and experimental spectra were in agreement after accounting for increases in the droplet sound speed with frequency. The agreement between experiment and model indicate that droplets (which have negligible optical absorption in the visible and infrared spectra by themselves) emitted pressure waves related to the droplet composition and size, and was independent of the physical characteristics of the optical absorbing nanoparticles. The diameter of individual droplets was calculated using three independent methods: the time domain photoacoustic signal, the time domain pulse echo ultrasound signal, and a fit to the photoacoustic model, then compared to the diameter as measured by optical microscopy. It was found the photoacoustic and ultrasound methods calculated diameters an average of 2.6% of each other, and 8.8% lower than that measured using optical microscopy. The discrepancy between the calculated diameters and the optical measurements may be due to the difficulty in resolving the droplet edges after being embedded in the translucent gelatin medium.

Review of combined isotopic and optical nanoscopy

PubMed Central

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

2017-01-01

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

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

PubMed Central

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

2018-01-01

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

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

NASA Astrophysics Data System (ADS)

Mulligan, Jeffrey A.; Adie, Steven G.

2017-02-01

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

Two-dimensional directional synthetic aperture focusing technique using acoustic-resolution photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Jeon, Seungwan; Park, Jihoon; Kim, Chulhong

2018-02-01

Photoacoustic microscopy (PAM) is a hybrid imaging technology using optical illumination and acoustic detection. PAM is divided into two types: optical-resolution PAM (OR-PAM) and acoustic-resolution photoacoustic microscopy (AR-PAM). Among them, AR-PAM has a great advantage in the penetration depth compared to OR-PAM because ARPAM relies on the acoustic focus, which is much less scattered in biological tissue than optical focus. However, because the acoustic focus is not as tight as the optical focus with a same numerical aperture (NA), the AR-PAM requires acoustic NA higher than optical NA. The high NA of the acoustic focus produces good image quality in the focal zone, but significantly degrades spatial resolution and signal-to-noise ratio (SNR) in the out-of-focal zone. To overcome the problem, synthetic aperture focusing technique (SAFT) has been introduced. SAFT improves the degraded image quality in terms of both SNR and spatial resolution in the out-of-focus zone by calculating the time delay of the corresponding signals and combining them. To extend the dimension of correction effect, several 2D SAFTs have been introduced, but there was a problem that the conventional 2D SAFTs cannot improve the degraded SNR and resolution as 1D SAFT can do. In this study, we proposed a new 2D SAFT that can compensate the distorted signals in x and y directions while maintaining the correction performance as the 1D SAFT.

Applicability of quantitative optical imaging techniques for intraoperative perfusion diagnostics: a comparison of laser speckle contrast imaging, sidestream dark-field microscopy, and optical coherence tomography

NASA Astrophysics Data System (ADS)

Jansen, Sanne M.; de Bruin, Daniel M.; Faber, Dirk J.; Dobbe, Iwan J. G. G.; Heeg, Erik; Milstein, Dan M. J.; Strackee, Simon D.; van Leeuwen, Ton G.

2017-08-01

Patient morbidity and mortality due to hemodynamic complications are a major problem in surgery. Optical techniques can image blood flow in real-time and high-resolution, thereby enabling perfusion monitoring intraoperatively. We tested the feasibility and validity of laser speckle contrast imaging (LSCI), optical coherence tomography (OCT), and sidestream dark-field microscopy (SDF) for perfusion diagnostics in a phantom model using whole blood. Microvessels with diameters of 50, 100, and 400 μm were constructed in a scattering phantom. Perfusion was simulated by pumping heparinized human whole blood at five velocities (0 to 20 mm/s). Vessel diameter and blood flow velocity were assessed with LSCI, OCT, and SDF. Quantification of vessel diameter was feasible with OCT and SDF. LSCI could only visualize the 400-μm vessel, perfusion units scaled nonlinearly with blood velocity. OCT could assess blood flow velocity in terms of inverse OCT speckle decorrelation time. SDF was not feasible to measure blood flow; however, for diluted blood the measurements were linear with the input velocity up to 1 mm/s. LSCI, OCT, and SDF were feasible to visualize blood flow. Validated blood flow velocity measurements intraoperatively in the desired parameter (mL·g-1) remain challenging.

Multifocal multiphoton microscopy with adaptive optical correction

NASA Astrophysics Data System (ADS)

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

2013-02-01

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

Coherent Femtosecond Spectroscopy and Nonlinear Optical Imaging on the Nanoscale

NASA Astrophysics Data System (ADS)

Kravtsov, Vasily

Optical properties of many materials and macroscopic systems are defined by ultrafast dynamics of electronic, vibrational, and spin excitations localized on the nanoscale. Harnessing these excitations for material engineering, optical computing, and control of chemical reactions has been a long-standing goal in science and technology. However, it is challenging due to the lack of spectroscopic techniques that can resolve processes simultaneously on the nanometer spatial and femtosecond temporal scales. This thesis describes the fundamental principles, implementation, and experimental demonstration of a novel type of ultrafast microscopy based on the concept of adiabatic plasmonic nanofocusing. Simultaneous spatio-temporal resolution on a nanometer-femtosecond scale is achieved by using a near-field nonlinear optical response induced by ultrafast surface plasmon polaritons nanofocused on a metal tip. First, we study the surface plasmon response in metallic structures and evaluate its prospects and limitations for ultrafast near-field microscopy. Through plasmon emission-based spectroscopy, we investigate dephasing times and interplay between radiative and non-radiative decay rates of localized plasmons and their modification due to coupling. We identify a new regime of quantum plasmonic coupling, which limits the achievable spatial resolution to several angstroms but at the same time provides a potential channel for generating ultrafast electron currents at optical frequencies. Next, we study propagation of femtosecond wavepackets of surface plasmon polaritons on a metal tip. In time-domain interferometric measurements we detect group delays that correspond to slowing of the plasmon polaritons down to 20% of the speed of light at the tip apex. This provides direct experimental verification of the plasmonic nanofocusing mechanism and suggests enhanced nonlinear optical interactions at the tip apex. We then measure a plasmon-generated third-order nonlinear optical four-wave mixing response from the tip apex and investigate its microscopic mechanism. Our results reveal a significant contribution to the third order nonlinearity of plasmonic structures due to large near-field gradients associated with nanofocused plasmons. In combination with scanning probe imaging and femtosecond pulse shaping, the nanofocused four-wave mixing response provides a basis for a novel type of ultrafast optical microscopy on the nanoscale. We demonstrate its capabilities by nano-imaging the coherent dynamics of localized plasmonic modes in a rough gold film edge with simultaneous sub-50 nm spatial and sub-5 fs temporal resolution. We capture the coherent decay and extract the dephasing times of individual plasmonic modes. Lastly, we apply our technique to study nanoscale spatial heterogeneity of the nonlinear optical response in novel two-dimensional materials: monolayer and few-layer graphene. An enhanced four-wave mixing signal is revealed on the edges of graphene flakes. We investigate the mechanism of this enhancement by performing nano-imaging on a graphene field-effect transistor with the variable carrier density controlled by electrostatic gating.

Aberrations and adaptive optics in super-resolution microscopy.

PubMed

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

2015-08-01

As one of the most powerful tools in the biological investigation of cellular structures and dynamic processes, fluorescence microscopy has undergone extraordinary developments in the past decades. The advent of super-resolution techniques has enabled fluorescence microscopy - or rather nanoscopy - to achieve nanoscale resolution in living specimens and unravelled the interior of cells with unprecedented detail. The methods employed in this expanding field of microscopy, however, are especially prone to the detrimental effects of optical aberrations. In this review, we discuss how super-resolution microscopy techniques based upon single-molecule switching, stimulated emission depletion and structured illumination each suffer from aberrations in different ways that are dependent upon intrinsic technical aspects. We discuss the use of adaptive optics as an effective means to overcome this problem. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy.

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

PubMed

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

2015-10-01

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

Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (RAMP) microscope.

PubMed

Otsu, Yo; Bormuth, Volker; Wong, Jerome; Mathieu, Benjamin; Dugué, Guillaume P; Feltz, Anne; Dieudonné, Stéphane

2008-08-30

Two-photon microscopy offers the promise of monitoring brain activity at multiple locations within intact tissue. However, serial sampling of voxels has been difficult to reconcile with millisecond timescales characteristic of neuronal activity. This is due to the conflicting constraints of scanning speed and signal amplitude. The recent use of acousto-optic deflector scanning to implement random-access multiphoton microscopy (RAMP) potentially allows to preserve long illumination dwell times while sampling multiple points-of-interest at high rates. However, the real-life abilities of RAMP microscopy regarding sensitivity and phototoxicity issues, which have so far impeded prolonged optical recordings at high frame rates, have not been assessed. Here, we describe the design, implementation and characterisation of an optimised RAMP microscope. We demonstrate the application of the microscope by monitoring calcium transients in Purkinje cells and cortical pyramidal cell dendrites and spines. We quantify the illumination constraints imposed by phototoxicity and show that stable continuous high-rate recordings can be obtained. During these recordings the fluorescence signal is large enough to detect spikes with a temporal resolution limited only by the calcium dye dynamics, improving upon previous techniques by at least an order of magnitude.

Correlated Time-Variation of Asphalt Rheology and Bulk Microstructure

NASA Astrophysics Data System (ADS)

Ramm, Adam; Nazmus, Sakib; Bhasin, Amit; Downer, Michael

We use noncontact optical microscopy and optical scattering in the visible and near-infrared spectrum on Performance Grade (PG) asphalt binder to confirm the existence of microstructures in the bulk. The number of visible microstructures increases linearly as penetration depth of the incident radiation increases, which verifies a uniform volume distribution of microstructures. We use dark field optical scatter in the near-infrared to measure the temperature dependent behavior of the bulk microstructures and compare this behavior with Dynamic Shear Rheometer (DSR) measurements of the bulk complex shear modulus | G* (T) | . The main findings are: (1) After reaching thermal equilibrium, both temperature dependent optical scatter intensity (I (T)) and bulk shear modulus (| G* (T) |) continue to change appreciably for times much greater than thermal equilibration times. (2) The hysteresis behavior during a complete temperature cycle seen in previous work derives from a larger time dependence in the cooling step compared with the heating step. (3) Different binder aging conditions show different thermal time-variations for both I (T) and | G* (T) | .

New ways of looking at very small holes - using optical nanoscopy to visualize liver sinusoidal endothelial cell fenestrations

NASA Astrophysics Data System (ADS)

Øie, Cristina I.; Mönkemöller, Viola; Hübner, Wolfgang; Schüttpelz, Mark; Mao, Hong; Ahluwalia, Balpreet S.; Huser, Thomas R.; McCourt, Peter

2018-02-01

Super-resolution fluorescence microscopy, also known as nanoscopy, has provided us with a glimpse of future impacts on cell biology. Far-field optical nanoscopy allows, for the first time, the study of sub-cellular nanoscale biological structures in living cells, which in the past was limited to electron microscopy (EM) (in fixed/dehydrated) cells or tissues. Nanoscopy has particular utility in the study of "fenestrations" - phospholipid transmembrane nanopores of 50-150 nm in diameter through liver sinusoidal endothelial cells (LSECs) that facilitate the passage of plasma, but (usually) not blood cells, to and from the surrounding hepatocytes. Previously, these fenestrations were only discernible with EM, but now they can be visualized in fixed and living cells using structured illumination microscopy (SIM) and in fixed cells using single molecule localization microscopy (SMLM) techniques such as direct stochastic optical reconstruction microscopy. Importantly, both methods use wet samples, avoiding dehydration artifacts. The use of nanoscopy can be extended to the in vitro study of fenestration dynamics, to address questions such as the following: are they actually dynamic structures, and how do they respond to endogenous and exogenous agents? A logical further extension of these methodologies to liver research (including the liver endothelium) will be their application to liver tissue sections from animal models with different pathological manifestations and ultimately to patient biopsies. This review will cover the current state of the art of the use of nanoscopy in the study of liver endothelium and the liver in general. Potential future applications in cell biology and the clinical implications will be discussed.

Optical nanoscopy to reveal structural and functional properties of liver cells (Presentation Recording)

NASA Astrophysics Data System (ADS)

McCourt, Peter; Huser, Thomas R.; Sørensen, Karen K.; Øie, Cristina I.; Mönkemöller, Viola; Ahluwalia, Balpreet S.

2015-08-01

The advent of optical nanoscopy has provided an opportunity to study fundamental properties of nanoscale biological functions, such as liver sinusoidal endothelial cells (LSEC) and their fenestrations. The fenestrations are nano-pores (50-200 nm) on the LSEC plasma membrane that allow free passage of molecules through cells. The fenestrated LSEC also hase a voracious appetite for waste molecules, viruses and nanoparticles. LSEC daily remove huge amounts of waste, nanoparticles and virus from the blood. Pharmaceuticals also need to pass through these fenestrations to be activated (e.g. cholesterol reducing statins) or detoxified by hepatocytes. And, when we age, our LSEC fenestrations become smaller and fewer. Today, we study these cells and structures using either conventional light microscopy on living cells, or high-resolution (but static) methods such as transmission and scanning electron microscopy on fixed (i.e. dead) tissue. Such methods, while very powerful, yield no real time information about the uptake of virus or nanoparticles, nor any information about fenestration dynamics. Therefore, to study LS-SEC, we are now using optical nanoscopy methods, and developing our own, to map their functions in 4 dimensions. Attaining this goal will shed new light on the cell biology of the liver and how it keeps us alive. This paper describes the challenges of studying LS-SEC with light microscopy, as well as current and potential solutions to this challenge using optical nanoscopy.

Surface plasmon resonance microscopy: achieving a quantitative optical response

PubMed Central

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

2016-01-01

Surface plasmon resonance (SPR) imaging allows real-time label-free imaging based on index of refraction, and changes in index of refraction at an interface. Optical parameter analysis is achieved by application of the Fresnel model to SPR data typically taken by an instrument in a prism based configuration. We carry out SPR imaging on a microscope by launching light into a sample, and collecting reflected light through a high numerical aperture microscope objective. The SPR microscope enables spatial resolution that approaches the diffraction limit, and has a dynamic range that allows detection of subnanometer to submicrometer changes in thickness of biological material at a surface. However, unambiguous quantitative interpretation of SPR changes using the microscope system could not be achieved using the Fresnel model because of polarization dependent attenuation and optical aberration that occurs in the high numerical aperture objective. To overcome this problem, we demonstrate a model to correct for polarization diattenuation and optical aberrations in the SPR data, and develop a procedure to calibrate reflectivity to index of refraction values. The calibration and correction strategy for quantitative analysis was validated by comparing the known indices of refraction of bulk materials with corrected SPR data interpreted with the Fresnel model. Subsequently, we applied our SPR microscopy method to evaluate the index of refraction for a series of polymer microspheres in aqueous media and validated the quality of the measurement with quantitative phase microscopy. PMID:27782542

Improved wavefront correction for coherent image restoration.

PubMed

Zelenka, Claudius; Koch, Reinhard

2017-08-07

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

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

PubMed Central

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

2016-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2003-05-01

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

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

Nonlinear Polarimetric Microscopy for Biomedical Imaging

NASA Astrophysics Data System (ADS)

Samim, Masood

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

Cadmium sulphide (CdS) thin films deposited by chemical bath deposition (CBD) and dip coating techniques—a comparative study

NASA Astrophysics Data System (ADS)

Khimani, Ankurkumar J.; Chaki, Sunil H.; Malek, Tasmira J.; Tailor, Jiten P.; Chauhan, Sanjaysinh M.; Deshpande, M. P.

2018-03-01

The CdS thin films were deposited on glass slide substrates by Chemical Bath Deposition and dip coating techniques. The films thickness variation with deposition time showed maximum films deposition at 35 min for both the films. The energy dispersive analysis of x-ray showed both the films to be stoichiometric. The x-ray diffraction analysis confirmed the films possess hexagonal crystal structure. The transmission electron, scanning electron and optical microscopy study showed the films deposition to be uniform. The selected area electron diffraction exhibited ring patterns stating the films to be polycrystalline in nature. The atomic force microscopy images showed surface formed of spherical grains, hills and valleys. The recorded optical absorbance spectra analysis revealed the films possess direct optical bandgap having values of 2.25 eV for CBD and 2.40 eV for dip coating. The refractive index (η), extinction coefficient (k), complex dielectric constant (ε) and optical conductivity (σ 0) variation with wavelength showed maximum photon absorption till the respective wavelengths corresponding to the optical bandgap energy values. The recorded photoluminescence spectra showed two emission peaks. All the obtained results have been discussed in details.

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

PubMed

Eliceiri, Kevin W

2004-08-01

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

Aberrations and adaptive optics in super-resolution microscopy

PubMed Central

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

2015-01-01

As one of the most powerful tools in the biological investigation of cellular structures and dynamic processes, fluorescence microscopy has undergone extraordinary developments in the past decades. The advent of super-resolution techniques has enabled fluorescence microscopy – or rather nanoscopy – to achieve nanoscale resolution in living specimens and unravelled the interior of cells with unprecedented detail. The methods employed in this expanding field of microscopy, however, are especially prone to the detrimental effects of optical aberrations. In this review, we discuss how super-resolution microscopy techniques based upon single-molecule switching, stimulated emission depletion and structured illumination each suffer from aberrations in different ways that are dependent upon intrinsic technical aspects. We discuss the use of adaptive optics as an effective means to overcome this problem. PMID:26124194

An integrated coherent anti-Stokes Raman scattering and multiphoton imaging technique for liver disease diagnosis

NASA Astrophysics Data System (ADS)

Lin, Jian; Lu, Fake; Zheng, Wei; Yu, Hanry; Sheppard, Colin; Huang, Zhiwei

2012-03-01

Liver steatosis and fibrosis are two prevalence liver diseases and may eventually develop into hepatocellular carcinoma (HCC) Due to their prevalence and severity, much work has been done to develop efficient diagnostic methods and therapies. Nonlinear optical microscopy has high sensitivity and chemical specificity for major biochemical compounds, making it a powerful tool for tissue imaging without staining. In this study, three nonlinear microscopy imaging modalities are applied to the study of liver diseases in a bile duct ligation rat modal. CARS shows the distributions of fats or lipids quantitatively across the tissue; SHG visualizes the collagens; and TPEF reveals the morphology of hepatic cells. The results clearly show the development of liver steatosis and fibrosis with time, and the hepatic fat and collagen fibrils are quantified. This study demonstrates the ability of multimodal nonlinear optical microscopy for liver disease diagnosis, and may provide new insights into the understanding of the mechanisms of steatosis/fibrosis transformations at the cellular and molecular levels.

Augmented microscopy with near-infrared fluorescence detection

NASA Astrophysics Data System (ADS)

Watson, Jeffrey R.; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G. Michael; Anton, Rein; Romanowski, Marek

2015-03-01

Near-infrared (NIR) fluorescence has become a frequently used intraoperative technique for image-guided surgical interventions. In procedures such as cerebral angiography, surgeons use the optical surgical microscope for the color view of the surgical field, and then switch to an electronic display for the NIR fluorescence images. However, the lack of stereoscopic, real-time, and on-site coregistration adds time and uncertainty to image-guided surgical procedures. To address these limitations, we developed the augmented microscope, whereby the electronically processed NIR fluorescence image is overlaid with the anatomical optical image in real-time within the optical path of the microscope. In vitro, the augmented microscope can detect and display indocyanine green (ICG) concentrations down to 94.5 nM, overlaid with the anatomical color image. We prepared polyacrylamide tissue phantoms with embedded polystyrene beads, yielding scattering properties similar to brain matter. In this model, 194 μM solution of ICG was detectable up to depths of 5 mm. ICG angiography was then performed in anesthetized rats. A dynamic process of ICG distribution in the vascular system overlaid with anatomical color images was observed and recorded. In summary, the augmented microscope demonstrates NIR fluorescence detection with superior real-time coregistration displayed within the ocular of the stereomicroscope. In comparison to other techniques, the augmented microscope retains full stereoscopic vision and optical controls including magnification and focus, camera capture, and multiuser access. Augmented microscopy may find application in surgeries where the use of traditional microscopes can be enhanced by contrast agents and image guided delivery of therapeutics, including oncology, neurosurgery, and ophthalmology.

Optical properties of transparent glass–ceramics containing lithium–mica nanocrystals: Crystallization effect

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

Khani, V.; Alizadeh, P., E-mail: p-alizadeh@modares.ac.ir; Shakeri, M.S.

2013-09-01

Graphical abstract: Optical properties of transparent Li{sub 2}O–MgO–Al{sub 2}O{sub 3}–SiO{sub 2}–F glasses containing lithium–mica nanocrystals are studied and crystallization condition has been evaluated and optimized to produce transparent glass–ceramics. Crystallization temperatures were determined by differential thermal analysis and crystalline phases were identified and quantified by X-ray diffraction. Scanning electron microscopy was used for morphological variations and UV–vis absorption spectroscopy for comparative analysis of transparency. In order to investigate the optical properties of transparent glass–ceramics, optical band gap, Fermi energy level and Urbach energy are calculated. The results of the investigation illustrate that band gap is reduced with increases in crystallizationmore » time and temperature. Enhanced orderliness in the arrangement of atoms might be regarded as possible reasons for the above changes. - Highlights: • The optimum temperature and time of crystallization were determined. • Li–mica nanocrystals with size of <30 nm were formed using a two-step heat-treatment. • Optical band gap and Fermi energy of nanocrystalline materials decreased with increasing of crystallization temperature and time. • Urbach band tailing was decreased with increasing of crystallization condition. - Abstract: Optical properties of transparent Li{sub 2}O–MgO–Al{sub 2}O{sub 3}–SiO{sub 2}–F glasses containing lithium–mica nanocrystals were studied. The crystallization condition of these glasses was evaluated and optimized to produce transparent glass–ceramics. Crystallization temperatures were determined by differential thermal analysis and crystalline phases were identified and quantified by X-ray diffraction. Scanning electron microscopy was used to detect morphological changes and UV–vis absorption spectroscopy was used for comparative analysis of transparency. In order to investigate the optical properties of the transparent glass–ceramics, optical band gap, Fermi energy level and Urbach energy were calculated. The results of the investigation illustrate that the band gap is reduced with increases in crystallization time and temperature. Enhanced orderliness in the arrangement of atoms might be regarded as possible reasons for the above changes.« less

Magneto-optical imaging of thin magnetic films using spins in diamond

NASA Astrophysics Data System (ADS)

Simpson, David A.; Tetienne, Jean-Philippe; McCoey, Julia M.; Ganesan, Kumaravelu; Hall, Liam T.; Petrou, Steven; Scholten, Robert E.; Hollenberg, Lloyd C. L.

2016-03-01

Imaging the fields of magnetic materials provides crucial insight into the physical and chemical processes surrounding magnetism, and has been a key ingredient in the spectacular development of magnetic data storage. Existing approaches using the magneto-optic Kerr effect, x-ray and electron microscopy have limitations that constrain further development, and there is increasing demand for imaging and characterisation of magnetic phenomena in real time with high spatial resolution. Here we show how the magneto-optical response of an array of negatively-charged nitrogen-vacancy spins in diamond can be used to image and map the sub-micron stray magnetic field patterns from thin ferromagnetic films. Using optically detected magnetic resonance, we demonstrate wide-field magnetic imaging over 100 × 100 μm2 with sub-micron spatial resolution at video frame rates, under ambient conditions. We demonstrate an all-optical spin relaxation contrast imaging approach which can image magnetic structures in the absence of an applied microwave field. Straightforward extensions promise imaging with sub-μT sensitivity and sub-optical spatial and millisecond temporal resolution. This work establishes practical diamond-based wide-field microscopy for rapid high-sensitivity characterisation and imaging of magnetic samples, with the capability for investigating magnetic phenomena such as domain wall and skyrmion dynamics and the spin Hall effect in metals.

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

PubMed

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

2014-01-01

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

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

PubMed Central

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

2014-01-01

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

Annexin-V/quantum dot probes for multimodal apoptosis monitoring in living cells: improving bioanalysis using electrochemistry

NASA Astrophysics Data System (ADS)

Montón, Helena; Parolo, Claudio; Aranda-Ramos, Antonio; Merkoçi, Arben; Nogués, Carme

2015-02-01

There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry.There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry. Electronic supplementary information (ESI) available: Optical microscopy images of apoptotic induced cell cultures at different times and negative control of flow cytometry. See DOI: 10.1039/c4nr07191c

Comparative Actions of Barbiturates Studied by Pollen Grain Germination.

ERIC Educational Resources Information Center

Kordan, Herbert A.; Mumford, Pauline M.

1979-01-01

Describes a simple experimental system whereby the comparative actions of long, medium, and short-acting barbiturates can be demonstrated in a relatively short period of time under optical microscopy using pollen grains as the biological test or assay system. (Author/HM)

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

PubMed Central

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

2012-01-01

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

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

PubMed Central

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

2015-01-01

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

Photoinduced force microscopy: A technique for hyperspectral nanochemical mapping

NASA Astrophysics Data System (ADS)

Murdick, Ryan A.; Morrison, William; Nowak, Derek; Albrecht, Thomas R.; Jahng, Junghoon; Park, Sung

2017-08-01

Advances in nanotechnology have intensified the need for tools that can characterize newly synthesized nanomaterials. A variety of techniques has recently been shown which combines atomic force microscopy (AFM) with optical illumination including tip-enhanced Raman spectroscopy (TERS), scattering-type scanning near-field optical microscopy (sSNOM), and photothermal induced resonance microscopy (PTIR). To varying degrees, these existing techniques enable optical spectroscopy with the nanoscale spatial resolution inherent to AFM, thereby providing nanochemical interrogation of a specimen. Here we discuss photoinduced force microscopy (PiFM), a recently developed technique for nanoscale optical spectroscopy that exploits image forces acting between an AFM tip and sample to detect wavelength-dependent polarization within the sample to generate absorption spectra. This approach enables ∼10 nm spatial resolution with spectra that show correlation with macroscopic optical absorption spectra. Unlike other techniques, PiFM achieves this high resolution with virtually no constraints on sample or substrate properties. The applicability of PiFM to a variety of archetypal systems is reported here, highlighting the potential of PiFM as a useful tool for a wide variety of industrial and academic investigations, including semiconducting nanoparticles, nanocellulose, block copolymers, and low dimensional systems, as well as chemical and morphological mixing at interfaces.

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

Coherent nonlinear optical imaging: beyond fluorescence microscopy.

PubMed

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

2011-01-01

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

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

PubMed Central

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

2013-01-01

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

A combined confocal and magnetic resonance microscope for biological studies

NASA Astrophysics Data System (ADS)

Majors, Paul D.; Minard, Kevin R.; Ackerman, Eric J.; Holtom, Gary R.; Hopkins, Derek F.; Parkinson, Christopher I.; Weber, Thomas J.; Wind, Robert A.

2002-12-01

Complementary data acquired with different microscopy techniques provide a basis for establishing a more comprehensive understanding of cell function in health and disease, particularly when results acquired with different methodologies can be correlated in time and space. In this article, a novel microscope is described for studying live cells simultaneously with both confocal scanning laser fluorescence optical microscopy and magnetic resonance microscopy. The various design considerations necessary for integrating these two complementary techniques are discussed, the layout and specifications of the instrument are given, and examples of confocal and magnetic resonance images of large frog cells and model tumor spheroids obtained with the compound microscope are presented.

Advanced SLMs for microscopy

NASA Astrophysics Data System (ADS)

Linnenberger, A.

2018-02-01

Wavefront shaping devices such as deformable mirrors, liquid crystal spatial light modulators (SLMs), and active lenses are of considerable interest in microscopy for aberration correction, volumetric imaging, and programmable excitation. Liquid crystal SLMs are high resolution phase modulators capable of creating complex phase profiles to reshape, or redirect light within a three-dimensional (3D) volume. Recent advances in Meadowlark Optics (MLO) SLMs reduce losses by increasing fill factor from 83.4% to 96%, and improving resolution from 512 x 512 pixels to 1920 x 1152 pixels while maintaining a liquid crystal response time of 300 Hz at 1064 nm. This paper summarizes new SLM capabilities, and benefits for microscopy.

Optical Coherence Tomography in Cancer Imaging

NASA Astrophysics Data System (ADS)

Nam, Ahhyun Stephanie; Vakoc, Benjamin; Blauvelt, David; Chico-Calero, Isabel

Investigations into the biology of cancer and novel cancer therapies rely on preclinical mouse models and traditional histological endpoints. Drawbacks of this approach include a limit in the number of time points for evaluation and an increased number of animals per study. This has motivated the use of intravital microscopy, which can provide longitudinal imaging of critical tumor parameters. Here, the capabilities of OCT as an intravital microscopy of the tumor microenvironment are summarized, and the state of OCT adoption into cancer research is summarized.

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

NASA Astrophysics Data System (ADS)

Mittal, Richa

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

Pure optical photoacoustic microscopy

PubMed Central

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

2011-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2016-12-01

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

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

PubMed Central

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

2009-01-01

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

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

PubMed

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

2007-09-03

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

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

PubMed Central

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

2011-01-01

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

4Pi Microscopy.

PubMed

Schmidt, Roman; Engelhardt, Johann; Lang, Marion

2013-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2011-02-01

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

Atomic force microscopy of orb-spider-web-silks to measure surface nanostructuring and evaluate silk fibers per strand

NASA Astrophysics Data System (ADS)

Kane, D. M.; Naidoo, N.; Staib, G. R.

2010-10-01

Atomic force microscopy (AFM) study is used to measure the surface topology and roughness of radial and capture spider silks on the micro- and nanoscale. This is done for silks of the orb weaver spider Argiope keyserlingi. Capture silk has a surface roughness that is five times less than that for radial silk. The capture silk has an equivalent flatness of λ /100 (5-6 nm deep surface features) as an optical surface. This is equivalent to a very highly polished optical surface. AFM does show the number of silk fibers that make up a silk thread but geometric distortion occurs during sample preparation. This prevented AFM from accurately measuring the silk topology on the microscale in this study.

High resolution and deep tissue imaging using a near infrared acoustic resolution photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Moothanchery, Mohesh; Sharma, Arunima; Periyasamy, Vijitha; Pramanik, Manojit

2018-02-01

It is always a great challenge for pure optical techniques to maintain good resolution and imaging depth at the same time. Photoacoustic imaging is an emerging technique which can overcome the limitation by pulsed light illumination and acoustic detection. Here, we report a Near Infrared Acoustic-Resolution Photoacoustic Microscopy (NIR-AR-PAM) systm with 30 MHz transducer and 1064 nm illumination which can achieve a lateral resolution of around 88 μm and imaging depth of 9.2 mm. Compared to visible light NIR beam can penetrate deeper in biological tissue due to weaker optical attenuation. In this work, we also demonstrated the in vivo imaging capabilty of NIRARPAM by near infrared detection of SLN with black ink as exogenous photoacoustic contrast agent in a rodent model.

Unconventional methods of imaging: computational microscopy and compact implementations

NASA Astrophysics Data System (ADS)

McLeod, Euan; Ozcan, Aydogan

2016-07-01

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

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

NASA Astrophysics Data System (ADS)

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

2004-08-01

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

Time-resolved correlative optical microscopy of charge-carrier transport, recombination, and space-charge fields in CdTe heterostructures

DOE PAGES

Kuciauskas, Darius; Myers, Thomas H.; Barnes, Teresa M.; ...

2017-02-20

From time- and spatially resolved optical measurements, we show that extended defects can have a large effect on the charge-carrier recombination in II-VI semiconductors. In CdTe double heterostructures grown by molecular beam epitaxy on the InSb (100)-orientation substrates, we characterized the extended defects and found that near stacking faults the space-charge field extends by 2-5 μm. Charge carriers drift (with the space-charge field strength of 730-1,360 V cm -1) and diffuse (with the mobility of 260 ± 30 cm 2 V -1 s -1) toward the extended defects, where the minority-carrier lifetime is reduced from 560 ns to 0.25 ns.more » Furthermore, the extended defects are nonradiative recombination sinks that affect areas significantly larger than the typical crystalline grains in II-VI solar cells. From the correlative time-resolved photoluminescence and second-harmonic generation microscopy data, we developed a band-diagram model that can be used to analyze the impact of extended defects on solar cells and other electronic devices.« less

In vivo time-serial evaluation of laser-induced choroidal neovascularization in rats simultaneously using photoacoustic microscopy and optical coherence tomography

NASA Astrophysics Data System (ADS)

Dai, Cuixia; Li, Lin; Liu, Wenlu; Wang, Fenghua; Zhou, Chuanqing

2018-02-01

Determination of the precise location and degree of condition of the Choroidal neovascularization (CNV) lesion is essential for diagnosation Neovascular age-related macular degeneration (AMD) and evaluation the efficacy of treatment. Given the complimentary contrast mechanisms of Photoacoustic microscopy (PAM) and Optical coherence tomography (OCT), the combination of PAM and OCT imaging could potentially provide much sensitive and specific detection of CNV. In this paper, we validated the opportunity to evaluate the information of laser-induced CNV and presented the in vivo time-serial evaluation of the CNV by simultaneously using PAM and OCT techniques. In vivo PAM and OCT examination was performed after laser photocoagulation applied to the rat fundus at days 1, 3, 5, 7, 14. Time-serial results showed that CNV in rats increased to its maximum at day 7 and decreased at day 14. Evolution of CNV information was given in PAM images with a high contrast and details of high axial resolution OCT images were simultaneously given to show the hyperreflective reaction progress.

Time-resolved correlative optical microscopy of charge-carrier transport, recombination, and space-charge fields in CdTe heterostructures

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

Kuciauskas, Darius; Myers, Thomas H.; Barnes, Teresa M.

From time- and spatially resolved optical measurements, we show that extended defects can have a large effect on the charge-carrier recombination in II-VI semiconductors. In CdTe double heterostructures grown by molecular beam epitaxy on the InSb (100)-orientation substrates, we characterized the extended defects and found that near stacking faults the space-charge field extends by 2-5 μm. Charge carriers drift (with the space-charge field strength of 730-1,360 V cm -1) and diffuse (with the mobility of 260 ± 30 cm 2 V -1 s -1) toward the extended defects, where the minority-carrier lifetime is reduced from 560 ns to 0.25 ns.more » Furthermore, the extended defects are nonradiative recombination sinks that affect areas significantly larger than the typical crystalline grains in II-VI solar cells. From the correlative time-resolved photoluminescence and second-harmonic generation microscopy data, we developed a band-diagram model that can be used to analyze the impact of extended defects on solar cells and other electronic devices.« less

Emerging fiber optic endomicroscopy technologies towards noninvasive real-time visualization of histology in situ

NASA Astrophysics Data System (ADS)

Xi, Jiefeng; Zhang, Yuying; Huo, Li; Chen, Yongping; Jabbour, Toufic; Li, Ming-Jun; Li, Xingde

2010-09-01

This paper reviews our recent developments of ultrathin fiber-optic endomicroscopy technologies for transforming high-resolution noninvasive optical imaging techniques to in vivo and clinical applications such as early disease detection and guidance of interventions. Specifically we describe an all-fiber-optic scanning endomicroscopy technology, which miniaturizes a conventional bench-top scanning laser microscope down to a flexible fiber-optic probe of a small footprint (i.e. ~2-2.5 mm in diameter), capable of performing two-photon fluorescence and second harmonic generation microscopy in real time. This technology aims to enable realtime visualization of histology in situ without the need for tissue removal. We will also present a balloon OCT endoscopy technology which permits high-resolution 3D imaging of the entire esophagus for detection of neoplasia, guidance of biopsy and assessment of therapeutic outcome. In addition we will discuss the development of functional polymeric fluorescent nanocapsules, which use only FAD approved materials and potentially enable fast track clinical translation of optical molecular imaging and targeted therapy.

Measuring Roughnesses Of Optical Surfaces

NASA Technical Reports Server (NTRS)

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

1994-01-01

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

Widely accessible method for superresolution fluorescence imaging of living systems

PubMed Central

Dedecker, Peter; Mo, Gary C. H.; Dertinger, Thomas; Zhang, Jin

2012-01-01

Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM)] require specialized equipment, whereas single-molecule–based approaches [e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM)] involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment, genetically encodable labels, and simple and rapid data acquisition, is capable of providing two- to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging. PMID:22711840

Nanodiamond Landmarks for Subcellular Multimodal Optical and Electron Imaging

PubMed Central

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

2013-01-01

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

The light-sheet microscopy revolution

NASA Astrophysics Data System (ADS)

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

2018-05-01

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

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

NASA Astrophysics Data System (ADS)

Gundlach, Heinz

1994-05-01

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

High-Resolution Intravital Microscopy

PubMed Central

Andresen, Volker; Pollok, Karolin; Rinnenthal, Jan-Leo; Oehme, Laura; Günther, Robert; Spiecker, Heinrich; Radbruch, Helena; Gerhard, Jenny; Sporbert, Anje; Cseresnyes, Zoltan; Hauser, Anja E.; Niesner, Raluca

2012-01-01

Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy - the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped-illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector. PMID:23251402

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

DOEpatents

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

2011-09-27

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

Optical properties of hydrothermally synthesized TGA-capped CdS nanoparticles: controlling crystalline size and phase

NASA Astrophysics Data System (ADS)

Tavakoli Banizi, Zoha; Seifi, Majid

2017-10-01

TGA-capped CdS nanoparticles were obtained in the presence of thioglycolic acid (TGA) as capping agent via a facile hydrothermal method at relatively low temperature and over a short duration. As-synthesized TGA-capped CdS nanoparticles were characterized by x-ray diffraction, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectra, photoluminescence spectroscopy, Ultraviolet-visible spectroscopy and energy-dispersive x-ray spectroscopy. The products had spherical shapes, although their crystalline size and phase was dependent on temperature and time of the reaction. Photoluminescence spectra showed that the fluorescence intensity decreased when increasing the reaction time and temperature.

Line-scan Raman microscopy complements optical coherence tomography for tumor boundary detection

NASA Astrophysics Data System (ADS)

Sudheendran, Narendran; Qi, Ji; Young, Eric D.; Lazar, Alexander J.; Lev, Dina C.; Pollock, Raphael E.; Larin, Kirill V.; Shih, Wei-Chuan

2014-10-01

Current technique for tumor resection requires biopsy of the tumor region and histological confirmation before the surgeon can be certain that the entire tumor has been resected. This confirmation process is time consuming both for the surgeon and the patient and also requires sacrifice of healthy tissue, motivating the development of novel technologies which can enable real-time detection of tumor-healthy tissue boundary for faster and more efficient surgeries. In this study, the potential of combining structural information from optical coherence tomography (OCT) and molecular information from line-scan Raman microscopy (LSRM) for such an application is presented. The results show a clear presence of boundary between myxoid liposarcoma and normal fat which is easily identifiable both from structural and molecular information. In cases where structural images are indistinguishable, for example, in normal fat and well differentiated liposarcoma (WDLS) or gastrointestinal sarcoma tumor (GIST) and myxoma, distinct molecular spectra have been obtained. The results suggest LSRM can effectively complement OCT to tumor boundary demarcation with high specificity.

High-speed transport-of-intensity phase microscopy with an electrically tunable lens.

PubMed

Zuo, Chao; Chen, Qian; Qu, Weijuan; Asundi, Anand

2013-10-07

We present a high-speed transport-of-intensity equation (TIE) quantitative phase microscopy technique, named TL-TIE, by combining an electrically tunable lens with a conventional transmission microscope. This permits the specimen at different focus position to be imaged in rapid succession, with constant magnification and no physically moving parts. The simplified image stack collection significantly reduces the acquisition time, allows for the diffraction-limited through-focus intensity stack collection at 15 frames per second, making dynamic TIE phase imaging possible. The technique is demonstrated by profiling of microlens array using optimal frequency selection scheme, and time-lapse imaging of live breast cancer cells by inversion the defocused phase optical transfer function to correct the phase blurring in traditional TIE. Experimental results illustrate its outstanding capability of the technique for quantitative phase imaging, through a simple, non-interferometric, high-speed, high-resolution, and unwrapping-free approach with prosperous applications in micro-optics, life sciences and bio-photonics.

Digital holographic microscopy long-term and real-time monitoring of cell division and changes under simulated zero gravity.

PubMed

Pan, Feng; Liu, Shuo; Wang, Zhe; Shang, Peng; Xiao, Wen

2012-05-07

The long-term and real-time monitoring the cell division and changes of osteoblasts under simulated zero gravity condition were succeed by combing a digital holographic microscopy (DHM) with a superconducting magnet (SM). The SM could generate different magnetic force fields in a cylindrical cavity, where the gravitational force of biological samples could be canceled at a special gravity position by a high magnetic force. Therefore the specimens were levitated and in a simulated zero gravity environment. The DHM was modified to fit with SM by using single mode optical fibers and a vertically-configured jig designed to hold specimens and integrate optical device in the magnet's bore. The results presented the first-phase images of living cells undergoing dynamic divisions and changes under simulated zero gravity environment for a period of 10 hours. The experiments demonstrated that the SM-compatible DHM setup could provide a highly efficient and versatile method for research on the effects of microgravity on biological samples.

Ultrafast recovery time and broadband saturable absorption properties of black phosphorus suspension

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

Wang, Yingwei; Huang, Guanghui; Chen, Jiazhang

2015-08-31

As a new type of two-dimensional crystal material, black phosphorus (BP) exhibits excellent electronics and optical performance. Herein, we focus on carrier relaxation dynamics and nonlinear optical properties of BP suspension. Atomic force microscopy, transmission electron microscopy, and optical transmission spectrum are employed to characterize the structure and linear optical properties of the BP. Additionally, pump-probe experiments at wavelength of 1550 nm were carried out to study the carrier dynamics in BP suspension, and ultrafast recovery time was observed (τ{sub s} = 24 ± 2 fs). Furthermore, we demonstrate the saturable absorption signals by open aperture Z-scan experiments at wavelengths of 1550 nm, 532 nm, and 680 nm. Themore » results indicate that BP has broadband saturable absorption properties and the nonlinear absorption coefficients were determined to be β{sub 2} = −0.20 ± 0.08 × 10{sup −3 }cm/GW (532 nm), β{sub 2} = −0.12 ± 0.05 × 10{sup −3 }cm/GW (680 nm), and β{sub 2} = −0.15 ± 0.09 × 10{sup −3 }cm/GW (1550 nm)« less

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

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

NASA Astrophysics Data System (ADS)

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

2006-05-01

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

Deformable Mirrors Correct Optical Distortions

NASA Technical Reports Server (NTRS)

2010-01-01

By combining the high sensitivity of space telescopes with revolutionary imaging technologies consisting primarily of adaptive optics, the Terrestrial Planet Finder is slated to have imaging power 100 times greater than the Hubble Space Telescope. To this end, Boston Micromachines Corporation, of Cambridge, Massachusetts, received Small Business Innovation Research (SBIR) contracts from the Jet Propulsion Laboratory for space-based adaptive optical technology. The work resulted in a microelectromechanical systems (MEMS) deformable mirror (DM) called the Kilo-DM. The company now offers a full line of MEMS DMs, which are being used in observatories across the world, in laser communication, and microscopy.

Advanced Fiber-optic Monitoring System for Space-flight Applications

NASA Technical Reports Server (NTRS)

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

2005-01-01

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

Quantitative PCR Coupled with Melt Curve Analysis for Detection of Selected Pseudo-nitzschia spp. (Bacillariophyceae) from the Northwestern Mediterranean Sea▿

PubMed Central

Andree, Karl B.; Fernández-Tejedor, Margarita; Elandaloussi, Laurence M.; Quijano-Scheggia, Sonia; Sampedro, Nagore; Garcés, Esther; Camp, Jordi; Diogène, Jorge

2011-01-01

The frequency and intensity of Pseudo-nitzschia spp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells of Pseudo-nitzschia from clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for all Pseudo-nitzschia spp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. Total Pseudo-nitzschia cell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eight Pseudo-nitzschia species-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the total Pseudo-nitzschia cell counts from optical microscopy. PMID:21193668

Synthesis of colloidal silver iron oxide nanoparticles--study of their optical and magnetic behavior.

PubMed

Kumar, Anil; Singhal, Aditi

2009-07-22

Silver iron oxide nanoparticles of fairly small size (average diameter approximately 1 nm) with narrow size distribution have been synthesized by the interaction of colloidal beta- Fe2O3 and silver nanoparticles. The surface morphology and size of these particles have been analyzed by using atomic force microscopy (AFM), field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). Their structural analysis has been carried out by employing x-ray diffraction (XRD), selected-area electron diffraction (SAED), optical and infrared (IR) spectroscopic techniques. The ageing of these particles exhibits the formation of self-assembly, possibly involving weak supramolecular interactions between Ag(I)O4 and Fe(III)O4 species. These particles display the onset of absorption in the near-infrared region and have higher absorption coefficient in the visible range compared to that of its precursors. Magnetic measurements reveal an interesting transition in their magnetic behavior from diamagnetic to superparamagnetic. The magnetic moment of these particles attains a limiting value of about 0.19 emu cm(-2), which is more than two times higher than that of colloidal beta- Fe2O3. With enhanced optical and magnetic properties, this system is suggested to have possible applications in optoelectronic and magnetic devices.

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

NASA Technical Reports Server (NTRS)

Vikram, Chandra S.; Witherow, William K.

2000-01-01

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

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.

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

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

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

2016-10-01

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

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

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

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

2016-03-30

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

A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

PubMed Central

Tam, Johnny; Cordier, Guillaume Alan; Bálint, Štefan; Sandoval Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike

2014-01-01

Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images. PMID:25545548

Multimodal non-linear optical imaging for the investigation of drug nano-/microcrystal-cell interactions.

PubMed

Darville, Nicolas; Saarinen, Jukka; Isomäki, Antti; Khriachtchev, Leonid; Cleeren, Dirk; Sterkens, Patrick; van Heerden, Marjolein; Annaert, Pieter; Peltonen, Leena; Santos, Hélder A; Strachan, Clare J; Van den Mooter, Guy

2015-10-01

Drug nano-/microcrystals are being used for sustained parenteral drug release, but safety and efficacy concerns persist as the knowledge of the in vivo fate of long-living particulates is limited. There is a need for techniques enabling the visualization of drug nano-/microcrystals in biological matrices. The aim of this work was to explore the potential of coherent anti-Stokes Raman scattering (CARS) microscopy, supported by other non-linear optical methods, as an emerging tool for the investigation of cellular and tissue interactions of unlabeled and non-fluorescent nano-/microcrystals. Raman and CARS spectra of the prodrug paliperidone palmitate (PP), paliperidone (PAL) and several suspension stabilizers were recorded. PP nano-/microcrystals were incubated with RAW 264.7 macrophages in vitro and their cellular disposition was investigated using a fully-integrated multimodal non-linear optical imaging platform. Suitable anti-Stokes shifts (CH stretching) were identified for selective CARS imaging. CARS microscopy was successfully applied for the selective three-dimensional, non-perturbative and real-time imaging of unlabeled PP nano-/microcrystals having dimensions larger than the optical lateral resolution of approximately 400nm, in relation to the cellular framework in cell cultures and ex vivo in histological sections. In conclusion, CARS microscopy enables the non-invasive and label-free imaging of (sub)micron-sized (pro-)drug crystals in complex biological matrices and could provide vital information on poorly understood nano-/microcrystal-cell interactions in future. Copyright © 2015 Elsevier B.V. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

Preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens and their use for bacterial detection.

PubMed

Dykman, Lev A; Staroverov, Sergei A; Guliy, Olga I; Ignatov, Oleg V; Fomin, Alexander S; Vidyasheva, Irina V; Karavaeva, Olga A; Bunin, Viktor D; Burygin, Gennady L

2012-01-01

This article reports the first preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens by using a combinatorial phage library of sheep antibodies. The prepared phage antibodies were used for the first time for lipopolysaccharide and flagellin detection by dot assay, electro-optical analysis of cell suspensions, and transmission electron microscopy. Interaction of A. brasilense Sp245 with antilipopolysaccharide and antiflagellin phage-displayed miniantibodies caused the magnitude of the electro-optical signal to change considerably. The electro-optical results were in good agreement with the electron microscopic data. This is the first reported possibility of employing phage-displayed miniantibodies in bacterial detection aided by electro-optical analysis of cell suspensions.

Analysis and Design of a Fiber-optic Probe for DNA Sensors Final Report CRADA No. TSB-1147-95

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

Molau, Nicole; Vail, Curtis

In 1995, a challenge in the field of genetics dealt with the acquisition of efficient DNA sequencing techniques for reading the 3 billion base-pairs that comprised the human genome. AccuPhotonics, Inc. proposed to develop and manufacture a state-of-the-art near-field scanning optical microscopy (NSOM) fiber-optic probe that was expected to increase probe efficiency by two orders of magnitude over the existing state-of-the-art and to improve resolution to 10Å. The detailed design calculation and optimization of electrical properties of the fiber-optic probe tip geometry would be performed at LLNL, using existing finite-difference time-domain (FDTD) electromagnetic (EM) codes.

Optical nonlinear absorption characteristics of Sb2Se3 nanoparticles

NASA Astrophysics Data System (ADS)

Muralikrishna, Molli; Kiran, Aditha Sai; Ravikanth, B.; Sowmendran, P.; Muthukumar, V. Sai; Venkataramaniah, Kamisetti

2014-04-01

In this work, we report for the first time, the nonlinear optical absorption properties of antimony selenide (Sb2Se3) nanoparticles synthesized through solvothermal route. X-ray diffraction results revealed the crystalline nature of the nanoparticles. Electron microscopy studies revealed that the nanoparticles are in the range of 10 - 40 nm. Elemental analysis was performed using EDAX. By employing open aperture z-scan technique, we have evaluated the effective two-photon absorption coefficient of Sb2Se3 nanoparticles to be 5e-10 m/W at 532 nm. These nanoparticles exhibit strong intensity dependent nonlinear optical absorption and hence could be considered to have optical power limiting applications in the visible range.

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

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

Hybrid Optical-Ultrasonic Technique for Biomedical Diagnostics

PubMed Central

Marcu, L.; Sun, Y.; Stephens, D.; Park, J.; Farwell, D. G.; Shung, K. K.

2010-01-01

We report the development of a diagnostic system combining time-resolved fluorescence spectroscopy and ultrasound backscatter microscopy and its application in diagnosis of tumors and atherosclerotic disease. This system allows for concurrent evaluation of distinct compositional, functional, and micro-anatomical features of normal and diseased tissues. PMID:21918737

High resolution multiple excitation spot optical microscopy

NASA Astrophysics Data System (ADS)

Dilipkumar, Shilpa; Mondal, Partha Pratim

2011-06-01

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

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

PubMed

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

2015-01-01

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

Imaging bio-distribution of a topically applied dermatological cream on minipig skin using fluorescence lifetime imaging microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Alex, Aneesh; Chaney, Eric J.; Criley, Jennifer M.; Spillman, Darold R.; Hutchison, Phaedra B.; Li, Joanne; Marjanovic, Marina; Frey, Steve; Cook, Steven; Boppart, Stephen A.; Arp, Zane A.

2017-02-01

Currently there is a lack of in vivo techniques to evaluate the spatial bio-distribution of dermal drugs over time without the need to take multiple serial biopsies. To address this gap, we investigated the use of multi-photon optical imaging methods to non-invasively track drug distribution on miniature pig (Species: Sus scrofa, Strain: Göttingen) skin in vivo. Minipig skin is the standard comparative research model to human skin, and is anatomically and functionally similar. We employed fluorescence lifetime imaging microscopy (FLIM) to visualize the spatial distribution and residency time of a topically applied experimental dermatological cream. This was made possible by the endogenous fluorescent optical properties of the experimental drug (fluorescence lifetime > 3000 ps). Two different drug formulations were applied on 2 minipigs for 7 consecutive days, with the control creams applied on the contralateral side, followed by 7 days of post-application monitoring using a multi-modal optical imaging system (MPTflex-CARS, JenLab, Germany). FLIM images were obtained from the treated regions 24 hr post-application from day 1 to day 14 that allowed visualization of cellular and sub-cellular features associated with different dermal layers non-invasively to a depth of 200 µm. Five punch biopsies per animal were obtained from the corresponding treated regions between days 8 and 14 for bioanalytical analysis and comparison with results obtained using FLIM. In conclusion, utilization of non-invasive optical biopsy methods for dermal drug evaluation can provide true longitudinal monitoring of drug spatial distribution, remove sampling limitations, and be more time-efficient compared to traditional methods.

Quantitative evaluation of morphological changes in activated platelets in vitro using digital holographic microscopy.

PubMed

Kitamura, Yutaka; Isobe, Kazushige; Kawabata, Hideo; Tsujino, Tetsuhiro; Watanabe, Taisuke; Nakamura, Masayuki; Toyoda, Toshihisa; Okudera, Hajime; Okuda, Kazuhiro; Nakata, Koh; Kawase, Tomoyuki

2018-06-18

Platelet activation and aggregation have been conventionally evaluated using an aggregometer. However, this method is suitable for short-term but not long-term quantitative evaluation of platelet aggregation, morphological changes, and/or adhesion to specific materials. The recently developed digital holographic microscopy (DHM) has enabled the quantitative evaluation of cell size and morphology without labeling or destruction. Thus, we aim to validate its applicability in quantitatively evaluating changes in cell morphology, especially in the aggregation and spreading of activated platelets, thus modifying typical image analysis procedures to suit aggregated platelets. Freshly prepared platelet-rich plasma was washed with phosphate-buffered saline and treated with 0.1% CaCl 2 . Platelets were then fixed and subjected to DHM, scanning electron microscopy (SEM), atomic force microscopy, optical microscopy, and flow cytometry (FCM). Tightly aggregated platelets were identified as single cells. Data obtained from time-course experiments were plotted two-dimensionally according to the average optical thickness versus attachment area and divided into four regions. The majority of the control platelets, which supposedly contained small and round platelets, were distributed in the lower left region. As activation time increased, however, this population dispersed toward the upper right region. The distribution shift demonstrated by DHM was essentially consistent with data obtained from SEM and FCM. Therefore, DHM was validated as a promising device for testing platelet function given that it allows for the quantitative evaluation of activation-dependent morphological changes in platelets. DHM technology will be applicable to the quality assurance of platelet concentrates, as well as diagnosis and drug discovery related to platelet functions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Characterization of TiN, TiC and Ti(C,N) in titanium-alloyed ferritic chromium steels focusing on the significance of different particle morphologies

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

Michelic, S.K., E-mail: susanne.michelic@unileoben.ac.at; Loder, D.; Reip, T.

2015-02-15

Titanium-alloyed ferritic chromium steels are a competitive option to classical austenitic stainless steels owing to their similar corrosion resistance. The addition of titanium significantly influences their final steel cleanliness. The present contribution focuses on the detailed metallographic characterization of titanium nitrides, titanium carbides and titanium carbonitrides with regard to their size, morphology and composition. The methods used are manual and automated Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy as well as optical microscopy. Additional thermodynamic calculations are performed to explain the precipitation procedure of the analyzed titanium nitrides. The analyses showed that homogeneous nucleation is decisive at an earlymore » process stage after the addition of titanium. Heterogeneous nucleation gets crucial with ongoing process time and essentially influences the final inclusion size of titanium nitrides. A detailed investigation of the nuclei for heterogeneous nucleation with automated Scanning Electron Microscopy proved to be difficult due to their small size. Manual Scanning Electron Microscopy and optical microscopy have to be applied. Furthermore, it was found that during solidification an additional layer around an existing titanium nitride can be formed which changes the final inclusion morphology significantly. These layers are also characterized in detail. Based on these different inclusion morphologies, in combination with thermodynamic results, tendencies regarding the formation and modification time of titanium containing inclusions in ferritic chromium steels are derived. - Graphical abstract: Display Omitted - Highlights: • The formation and modification of TiN in the steel 1.4520 was examined. • Heterogeneous nucleation essentially influences the final steel cleanliness. • In most cases heterogeneous nuclei in TiN inclusions are magnesium based. • Particle morphology provides important information on inclusion formation.« less

Combined Optical Coherence and Fluorescence Microscopy to assess dynamics and specificity of pancreatic beta-cell tracers

PubMed Central

Berclaz, Corinne; Pache, Christophe; Bouwens, Arno; Szlag, Daniel; Lopez, Antonio; Joosten, Lieke; Ekim, Selen; Brom, Maarten; Gotthardt, Martin; Grapin-Botton, Anne; Lasser, Theo

2015-01-01

The identification of a beta-cell tracer is a major quest in diabetes research. However, since MRI, PET and SPECT cannot resolve individual islets, optical techniques are required to assess the specificity of these tracers. We propose to combine Optical Coherence Microscopy (OCM) with fluorescence detection in a single optical platform to facilitate these initial screening steps from cell culture up to living rodents. OCM can image islets and vascularization without any labeling. Thereby, it alleviates the need of both genetically modified mice to detect islets and injection of external dye to reveal vascularization. We characterized Cy5.5-exendin-3, an agonist of glucagon-like peptide 1 receptor (GLP1R), for which other imaging modalities have been used and can serve as a reference. Cultured cells transfected with GLP1R and incubated with Cy5.5-exendin-3 show full tracer internalization. We determined that a dose of 1 μg of Cy5.5-exendin-3 is sufficient to optically detect in vivo the tracer in islets with a high specificity. In a next step, time-lapse OCM imaging was used to monitor the rapid and specific tracer accumulation in murine islets and its persistence over hours. This optical platform represents a versatile toolbox for selecting beta-cell specific markers for diabetes research and future clinical diagnosis. PMID:25988507

Techniques for super-resolution microscopy using NV-diamond

NASA Astrophysics Data System (ADS)

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

2011-05-01

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

Value of Reflected Light Microscopy in Teaching.

ERIC Educational Resources Information Center

Pasteris, Jill Dill

1983-01-01

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

In situ monitoring of atomic layer epitaxy via optical ellipsometry

NASA Astrophysics Data System (ADS)

Lyzwa, F.; Marsik, P.; Roddatis, V.; Bernhard, C.; Jungbauer, M.; Moshnyaga, V.

2018-03-01

We report on the use of time-resolved optical ellipsometry to monitor the deposition of single atomic layers with subatomic sensitivity. Ruddlesden-Popper thin films of SrO(SrTiO3) n=4 were grown by means of metalorganic aerosol deposition in the atomic layer epitaxy mode on SrTiO3(1 0 0), LSAT(1 0 0) and DyScO3(1 1 0) substrates. The measured time dependences of ellipsometric angles, Δ(t) and Ψ(t), were described by using a simple optical model, considering the sequence of atomic layers SrO and TiO2 with corresponding bulk refractive indices. As a result, valuable online information on the atomic layer epitaxy process was obtained. Ex situ characterization techniques, i.e. transmission electron microscopy, x-ray diffraction and x-ray reflectometry verify the crystal structure and confirm the predictions of optical ellipsometry.

Structural and optical properties of PbS thin films grown by chemical bath deposition

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

Seghaier, S.; Kamoun, N.; Guasch, C.

2007-09-19

Lead sulphide thin films are grown on glass substrates at various deposition times tD, in the range of 40-60 min per step of 2 min, using the chemical bath deposition technique. X-ray diffraction and atomic force microscopy are used to characterize the film structure. The surface composition is analysed by Auger electron spectroscopy. It appears that the as-prepared thin films are polycrystalline with cubic structure. Nanometric scale crystallites are uniformly distributed on the surface. They exhibit almost a stoechiometric composition with a [Pb]/[S] ratio equal to 1.10. Optical properties are studied in the range of 300-3300 nm by spectrophotometric measurements.more » Analysis of the optical absorption data of lead sulphide thin layers reveals a narrow optical direct band gap equal to 0.46 eV for the layer corresponding to a deposition time equal to 60 min.« less

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

NASA Astrophysics Data System (ADS)

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

2006-02-01

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

Structural and spectroscopic analysis of ex-situ annealed RF sputtered aluminium doped zinc oxide thin films

NASA Astrophysics Data System (ADS)

Otieno, Francis; Airo, Mildred; Erasmus, Rudolph M.; Billing, David G.; Quandt, Alexander; Wamwangi, Daniel

2017-08-01

Aluminium doped zinc oxide thin films are prepared by Radio Frequency magnetron sputtering in pure argon atmosphere at 100 W. The structural results reveal good film adhesion on a silicon substrate (001). The thin films were then subjected to heat treatment in a furnace under ambient air. The structural, morphological, and optical properties of the thin films as a function of deposition time and annealing temperatures have been investigated using Grazing incidence X-Ray Diffraction (GIXRD), Atomic Force Microscopy, and Scanning Electronic Microscopy. The photoluminescence properties of the annealed films showed significant changes in the optical properties attributed to mid gap defects. Annealing increases the crystallite size and the roughness of the film. The crystallinity of the films also improved as evident from the Raman and XRD studies.

Characterization of CuCl quantum dots grown in NaCl single crystals via optical measurements, X-ray diffraction, and transmission electron microscopy

NASA Astrophysics Data System (ADS)

Miyajima, Kensuke; Akatsu, Tatsuro; Itoh, Ken

2018-05-01

We evaluated the crystal size, shape, and alignment of the lattice planes of CuCl quantum dots (QDs) embedded in NaCl single crystals by optical measurements, X-ray diffraction (XRD) patterns, and transmission electron microscopy (TEM). We obtained, for the first time, an XRD pattern and TEM images for CuCl QDs in NaCl crystals. The XRD pattern showed that the lattice planes of the CuCl QDs were parallel to those of the NaCl crystals. In addition, the size of the QDs was estimated from the diffraction width. It was apparent from the TEM images that almost all CuCl QDs were polygonal, although some cubic QDs were present. The mean size and size distribution of the QDs were also obtained. The dot size obtained from optical measurements, XRD, and TEM image were almost consistent. Our new findings can help to reveal the growth mechanism of semiconductor QDs embedded in a crystallite matrix. In addition, this work will play an important role in progressing the study of optical phenomena originating from assembled semiconductor QDs.

Ultrathin forward-imaging short multimode fiber probe for full-field optical coherence microscopy

NASA Astrophysics Data System (ADS)

Sato, Manabu; Saito, Daisuke; Shouji, Kou; Kurotani, Reiko; Abe, Hiroyuki; Nishidate, Izumi

2016-12-01

To extend the applications of optical coherence tomography (OCT) to the fields of physiology and clinical medicine, less invasive, robust, and reliable optical probes are required. Thus, we demonstrate an ultrathin forward-imaging short multimode fiber (SMMF) optical coherence microscopy (OCM) probe with a 50 μm core diameter, 125 μm total diameter, and 5.12 mm length. Imaging conditions and magnification were analyzed, and they correspond closely to the measured results. The dispersion of the SMMF was investigated, and the modal dispersion coefficient was found to be 2.3% of the material dispersion coefficient. The axial resolution was minimized at 2.15 μm using a 0.885-mm-thick dispersion compensator. The lateral resolution was evaluated to be 4.38 μm using a test pattern. The contrast of the OCM images was 5.7 times higher than that of the signal images owing to the coherence gate. The depth of focus and diameter of the field of view were measured to be 60 μm and 40-50 μm, respectively. OCM images of the dried fins of small fish (Medaka) were measured and internal structures could be recognized.

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.

Atomic force-multi-optical imaging integrated microscope for monitoring molecular dynamics in live cells.

PubMed

Trache, Andreea; Meininger, Gerald A

2005-01-01

A novel hybrid imaging system is constructed integrating atomic force microscopy (AFM) with a combination of optical imaging techniques that offer high spatial resolution. The main application of this instrument (the NanoFluor microscope) is the study of mechanotransduction with an emphasis on extracellular matrix-integrin-cytoskeletal interactions and their role in the cellular responses to changes in external chemical and mechanical factors. The AFM allows the quantitative assessment of cytoskeletal changes, binding probability, adhesion forces, and micromechanical properties of the cells, while the optical imaging applications allow thin sectioning of the cell body at the coverslip-cell interface, permitting the study of focal adhesions using total internal reflection fluorescence (TIRF) and internal reflection microscopy (IRM). Combined AFM-optical imaging experiments show that mechanical stimulation at the apical surface of cells induces a force-generating cytoskeletal response, resulting in focal contact reorganization on the basal surface that can be monitored in real time. The NanoFluor system is also equipped with a novel mechanically aligned dual camera acquisition system for synthesized Forster resonance energy transfer (FRET). The integrated NanoFluor microscope system is described, including its characteristics, applications, and limitations.

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

NASA Astrophysics Data System (ADS)

Suen, Ricky Wai

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

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

PubMed

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

2015-09-01

Multiconfocal microscopy gives a good compromise between fast imaging and reasonable resolution. However, the low intensity of live fluorescent emitters is a major limitation to this technique. Aberrations induced by the optical setup, especially the mismatch of the refractive index and the biological sample itself, distort the point spread function and further reduce the amount of detected photons. Altogether, this leads to impaired image quality, preventing accurate analysis of molecular processes in biological samples and imaging deep in the sample. The amount of detected fluorescence can be improved with adaptive optics. Here, we used a compact adaptive optics module (adaptive optics box for sectioning optical microscopy), which was specifically designed for spinning disk confocal microscopy. The module overcomes undesired anomalies by correcting for most of the aberrations in confocal imaging. Existing aberration detection methods require prior illumination, which bleaches the sample. To avoid multiple exposures of the sample, we established an experimental model describing the depth dependence of major aberrations. This model allows us to correct for those aberrations when performing a z-stack, gradually increasing the amplitude of the correction with depth. It does not require illumination of the sample for aberration detection, thus minimizing photobleaching and phototoxicity. With this model, we improved both signal-to-background ratio and image contrast. Here, we present comparative studies on a variety of biological samples. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Boundary segmentation for fluorescence microscopy using steerable filters

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

A submersible digital in-line holographic microscope

NASA Astrophysics Data System (ADS)

Jericho, Manfred; Jericho, Stefan; Kreuzer, Hans Juergen; Garcia, Jeorge; Klages, Peter

Few instruments exist that can image microscopic marine organisms in their natural environment so that their locomotion mechanisms, feeding habits and interactions with surfaces, such as bio-fouling, can be investigated in situ. In conventional optical microscopy under conditions of high magnification, only objects confined to the narrow focal plane can be imaged and processes that involve translation of the object perpendicular to this plane are not accessible. To overcome this severe limitation of optical microscopy, we developed digital in-line holographic microscopy (DIHM) as a high-resolution tool for the tracking of organisms in three dimensions. We describe here the design and performance of a very simple submersible digital in-line holographic microscope (SDIHM) that can image organisms and their motion with micron resolution and that can be deployed from small vessels. Holograms and reconstructed images of several microscopic marine organisms were successfully obtained down to a depth of 20 m. The maximum depth was limited by the length of data transmission cables available at the time and operating depth in excess of 100 m are easily possible for the instrument.

(Gene sequencing by scanning molecular exciton microscopy)

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

Not Available

1991-01-01

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

Correlated time-variation of bulk microstructure and rheology in asphalt binders.

PubMed

Ramm, A; Sakib, N; Bhasin, A; Downer, M C

2018-05-22

We use near-infrared dark-field optical microscopy to probe isothermal time variation of the volume fraction of naturally-occurring, subsurface microstructures in PG 64-22 asphalt binders at temperature T=30∘C, following a rapid heating (cooling) increment |ΔT|=20∘C from initial temperature T0=10∘C(50∘C). We compare these microstructure variations with isothermal time variations of the magnitude |G30∗(t)| of the bulk complex shear modulus measured for identical sample conditions with a Dynamic Shear Rheometer. The main findings are: (1) Microstructure volume fraction (inferred from intensity I(t) of near-infrared optical scatter) and |G∗(t)| both continue to change appreciably long after measurable changes of binder temperature cease. Moreover, delayed time variations in I(t) and |G∗(t)| (2) correlate closely with each other; (3) evolve on three distinct time scales - several minutes, ∼1 h, >1 day; (4) depend on binder aging; (5) are more pronounced after a cooling step (ΔT=-20∘C) than after a heating step (ΔT=+20∘C); and (6) account for hysteresis in I(t) and |G∗(t)| curves observed during heating-cooling cycles. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Temporal overlap estimation based on interference spectrum in CARS microscopy

NASA Astrophysics Data System (ADS)

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

2018-01-01

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

Diving under a microscope--a new simple and versatile in vitro diving device for fluorescence and confocal microscopy allowing the controls of hydrostatic pressure, gas pressures, and kinetics of gas saturation.

PubMed

Wang, Qiong; Belhomme, Marc; Guerrero, François; Mazur, Aleksandra; Lambrechts, Kate; Theron, Michaël

2013-06-01

How underwater diving effects the function of the arterial wall and the activities of endothelial cells is the focus of recent studies on decompression sickness. Here we describe an in vitro diving system constructed to achieve real-time monitoring of cell activity during simulated dives under fluorescent microscopy and confocal microscopy. A 1-mL chamber with sapphire windows on both sides and located on the stage of an inverted microscope was built to allow in vitro diving simulation of isolated cells or arteries in which activities during diving are monitored in real-time via fluorescent microscopy and confocal microscopy. Speed of compression and decompression can range from 20 to 2000 kPa/min, allowing systemic pressure to range up to 6500 kPa. Diving temperature is controlled at 37°C. During air dive simulation oxygen partial pressure is optically monitored. Perfusion speed can range from 0.05 to 10 mL/min. The system can support physiological viability of in vitro samples for real-time monitoring of cellular activity during diving. It allows regulations of pressure, speeds of compression and decompression, temperature, gas saturation, and perfusion speed. It will be a valuable tool for hyperbaric research.

A compact multi-trap optical tweezer system based on CD-ROM technologies

NASA Astrophysics Data System (ADS)

McMenamin, T.; Lee, W. M.

2017-08-01

We implemented an integrated time sharing multiple optical trapping system through the synchronisation of high speed voice coil scanning lens and laser pulsing. The integration is achieved by using commonly available optical pickup unit (OPU) that exists inside optical drives. Scanning frequencies of up to 2 kHz were showed to achieve arbitrary distribution of optical traps within the one-dimensional scan range of the voice coil motor. The functions of the system were demonstrated by the imaging and trapping of 1 μm particles and giant unilamellar vesicles (GUVs). The new device circumvents existing bulky laser scanning systems (4f lens systems) with an integrated laser and lens steering platform that can be integrated on a variety of microscopy platforms (confocal, lightsheet, darkfield).

Digitally switchable multi-focal lens using freeform optics.

PubMed

Wang, Xuan; Qin, Yi; Hua, Hong; Lee, Yun-Han; Wu, Shin-Tson

2018-04-16

Optical technologies offering electrically tunable optical power have found a broad range of applications, from head-mounted displays for virtual and augmented reality applications to microscopy. In this paper, we present a novel design and prototype of a digitally switchable multi-focal lens (MFL) that offers the capability of rapidly switching the optical power of the system among multiple foci. It consists of a freeform singlet and a customized programmable optical shutter array (POSA). Time-multiplexed multiple foci can be obtained by electrically controlling the POSA to switch the light path through different segments of the freeform singlet rapidly. While this method can be applied to a broad range of imaging and display systems, we experimentally demonstrate a proof-of-concept prototype for a multi-foci imaging system.

A novel graphene oxide-polyimide as optical waveguide material: Synthesis and thermo-optic switch properties

NASA Astrophysics Data System (ADS)

Cao, Tianlin; Zhao, Fanyu; Da, Zulin; Qiu, Fengxian; Yang, Dongya; Guan, Yijun; Cao, Guorong; Zhao, Zerun; Li, Jiaxin; Guo, Xiaotong

2016-10-01

In this work, a novel graphene oxide-polyimide (GOPI) as optical waveguide material was prepared. The structure, mechanical, thermal property and morphology of the GOPI was characterized by using fourier transform infrared, UV-visible spectroscopy, near-infrared spectrum, thermogravimetric analysis, differential scanning calorimetry, scanning electron microscope and transmission electron microscopy. The thermo-optic coefficients (dn/dT) are -9.16 × 10-4 (532 nm), -7.56 × 10-4 (650 nm) and -4.82 × 10-4 (850 nm) °C-1, respectively. Based on the thermo-optic effect of prepared GOPI as waveguide material, a Y-branch with branching angle of 0.143° and Mach-Zehnder thermo-optic switches were designed. Using finite difference beam propagation method (FD-BPM) method, the simulation results such as power consumptions and response times of two different thermo-optic switches were obtained.

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

PubMed

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

2006-06-30

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

Practical guidelines for implementing adaptive optics in fluorescence microscopy

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

SNSMIL, a real-time single molecule identification and localization algorithm for super-resolution fluorescence microscopy

PubMed Central

Tang, Yunqing; Dai, Luru; Zhang, Xiaoming; Li, Junbai; Hendriks, Johnny; Fan, Xiaoming; Gruteser, Nadine; Meisenberg, Annika; Baumann, Arnd; Katranidis, Alexandros; Gensch, Thomas

2015-01-01

Single molecule localization based super-resolution fluorescence microscopy offers significantly higher spatial resolution than predicted by Abbe’s resolution limit for far field optical microscopy. Such super-resolution images are reconstructed from wide-field or total internal reflection single molecule fluorescence recordings. Discrimination between emission of single fluorescent molecules and background noise fluctuations remains a great challenge in current data analysis. Here we present a real-time, and robust single molecule identification and localization algorithm, SNSMIL (Shot Noise based Single Molecule Identification and Localization). This algorithm is based on the intrinsic nature of noise, i.e., its Poisson or shot noise characteristics and a new identification criterion, QSNSMIL, is defined. SNSMIL improves the identification accuracy of single fluorescent molecules in experimental or simulated datasets with high and inhomogeneous background. The implementation of SNSMIL relies on a graphics processing unit (GPU), making real-time analysis feasible as shown for real experimental and simulated datasets. PMID:26098742

Axial range of conjugate adaptive optics in two-photon microscopy

PubMed Central

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

2015-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2008-02-01

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

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

PubMed

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

2011-06-08

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

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

PubMed

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

2015-08-10

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

Tutorial on photoacoustic tomography

NASA Astrophysics Data System (ADS)

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

2016-06-01

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

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

PubMed

Fu, Ling; Gu, Min

2006-05-15

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

Multimodal hyperspectral optical microscopy

DOE PAGES

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

2017-09-02

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

Multimodal hyperspectral optical microscopy

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

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

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

In vivo vascular flow profiling combined with optical tweezers based blood routing

NASA Astrophysics Data System (ADS)

Meissner, Robert; Sugden, Wade W.; Siekmann, Arndt F.; Denz, Cornelia

2017-07-01

In vivo wall shear rate is quantified during zebrafish development using particle image velocimetry for biomedical diagnosis and modeling of artificial vessels. By using brightfield microscopy based high speed video tracking we can resolve single heart-beat cycles of blood flow in both space and time. Maximum blood flow velocities and wall shear rates are presented for zebrafish at two and three days post fertilization. By applying biocompatible optical tweezers as an Optical rail we present rerouting of red blood cells in vivo. With purely light-driven means we are able to compensate the lack of proper red blood cell blood flow in so far unperfused capillaries.

Functional photoacoustic microscopy of pH

NASA Astrophysics Data System (ADS)

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

2012-02-01

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

Invited Review Article: Pump-probe microscopy.

PubMed

Fischer, Martin C; Wilson, Jesse W; Robles, Francisco E; Warren, Warren S

2016-03-01

Multiphoton microscopy has rapidly gained popularity in biomedical imaging and materials science because of its ability to provide three-dimensional images at high spatial and temporal resolution even in optically scattering environments. Currently the majority of commercial and home-built devices are based on two-photon fluorescence and harmonic generation contrast. These two contrast mechanisms are relatively easy to measure but can access only a limited range of endogenous targets. Recent developments in fast laser pulse generation, pulse shaping, and detection technology have made accessible a wide range of optical contrasts that utilize multiple pulses of different colors. Molecular excitation with multiple pulses offers a large number of adjustable parameters. For example, in two-pulse pump-probe microscopy, one can vary the wavelength of each excitation pulse, the detection wavelength, the timing between the excitation pulses, and the detection gating window after excitation. Such a large parameter space can provide much greater molecular specificity than existing single-color techniques and allow for structural and functional imaging without the need for exogenous dyes and labels, which might interfere with the system under study. In this review, we provide a tutorial overview, covering principles of pump-probe microscopy and experimental setup, challenges associated with signal detection and data processing, and an overview of applications.

Invited Review Article: Pump-probe microscopy

PubMed Central

Wilson, Jesse W.; Robles, Francisco E.; Warren, Warren S.

2016-01-01

Multiphoton microscopy has rapidly gained popularity in biomedical imaging and materials science because of its ability to provide three-dimensional images at high spatial and temporal resolution even in optically scattering environments. Currently the majority of commercial and home-built devices are based on two-photon fluorescence and harmonic generation contrast. These two contrast mechanisms are relatively easy to measure but can access only a limited range of endogenous targets. Recent developments in fast laser pulse generation, pulse shaping, and detection technology have made accessible a wide range of optical contrasts that utilize multiple pulses of different colors. Molecular excitation with multiple pulses offers a large number of adjustable parameters. For example, in two-pulse pump-probe microscopy, one can vary the wavelength of each excitation pulse, the detection wavelength, the timing between the excitation pulses, and the detection gating window after excitation. Such a large parameter space can provide much greater molecular specificity than existing single-color techniques and allow for structural and functional imaging without the need for exogenous dyes and labels, which might interfere with the system under study. In this review, we provide a tutorial overview, covering principles of pump-probe microscopy and experimental setup, challenges associated with signal detection and data processing, and an overview of applications. PMID:27036751

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

NASA Astrophysics Data System (ADS)

Ryseva, Ekaterina; Zhukova, Ekaterina

2013-05-01

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

Incorporating Basic Optical Microscopy in the Instrumental Analysis Laboratory

ERIC Educational Resources Information Center

Flowers, Paul A.

2011-01-01

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

Numerical modeling of two-photon focal modulation microscopy with a sinusoidal phase filter.

PubMed

Chen, Rui; Shen, Shuhao; Chen, Nanguang

2018-05-01

A spatiotemporal phase modulator (STPM) is theoretically investigated using the vectorial diffraction theory. The STPM is equivalent to a time-dependent phase-only pupil filter that alternates between a homogeneous filter and a stripe-shaped filter with a sinusoidal phase distribution. It is found that two-photon focal modulation microscopy (TPFMM) using this STPM can significantly suppress the background contribution from out-of-focus ballistic excitation and achieve almost the same resolution as two-photon microscopy. The modulation depth is also evaluated and a compromise exists between the signal-to-background ratio and signal-to-noise ratio. The theoretical investigations provide important insights into future implementations of TPFMM and its potential to further extend the penetration depth of nonlinear microscopy in imaging multiple-scattering biological tissues. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

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

PubMed

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

2017-12-15

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

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

PubMed

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

2015-07-01

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

Microscopy of biological sample through advanced diffractive optics from visible to X-ray wavelength regime.

PubMed

Di Fabrizio, Enzo; Cojoc, Dan; Emiliani, Valentina; Cabrini, Stefano; Coppey-Moisan, Maite; Ferrari, Enrico; Garbin, Valeria; Altissimo, Matteo

2004-11-01

The aim of this report is to demonstrate a unified version of microscopy through the use of advanced diffractive optics. The unified scheme derives from the technical possibility of realizing front wave engineering in a wide range of electromagnetic spectrum. The unified treatment is realized through the design and nanofabrication of phase diffractive elements (PDE) through which wave front beam shaping is obtained. In particular, we will show applications, by using biological samples, ranging from micromanipulation using optical tweezers to X-ray differential interference contrast (DIC) microscopy combined with X-ray fluorescence. We report some details on the design and physical implementation of diffractive elements that besides focusing also perform other optical functions: beam splitting, beam intensity, and phase redistribution or mode conversion. Laser beam splitting is used for multiple trapping and independent manipulation of micro-beads surrounding a cell as an array of tweezers and for arraying and sorting microscopic size biological samples. Another application is the Gauss to Laguerre-Gauss mode conversion, which allows for trapping and transfering orbital angular momentum of light to micro-particles immersed in a fluid. These experiments are performed in an inverted optical microscope coupled with an infrared laser beam and a spatial light modulator for diffractive optics implementation. High-resolution optics, fabricated by means of e-beam lithography, are demonstrated to control the intensity and the phase of the sheared beams in x-ray DIC microscopy. DIC experiments with phase objects reveal a dramatic increase in image contrast compared to bright-field x-ray microscopy. Besides the topographic information, fluorescence allows detection of certain chemical elements (Cl, P, Sc, K) in the same setup, by changing the photon energy of the x-ray beam. (c) 2005 Wiley-Liss, Inc.

Modeling of structure and properties of thermo-optical converters for laser surgery

NASA Astrophysics Data System (ADS)

Belikov, Andrey V.; Skrypnik, Alexei V.; Kurnyshev, Vadim Y.

2016-04-01

Volumetric fiber-optic thermal converter (VFOTC) formed on the end of the quartz fiber as a result of two-stage conversion of quartz and carbon by medical diode laser radiation with a wavelength of 980 nm is investigated both experimentally and theoretically. The geometrical dimensions of the converter are defined and the internal structure of the converter is studied by optical microscopy. The dependence of VFOTC temperature on exposure time of diode laser radiation with a wavelength of 980 nm and power of 1.0+/-0.1 W is obtained experimentally. The structural, optical and thermal model of VFOTC is proposed. Good correlation between the experimental and modeling results of laser heating of the converter is demonstrated.

Digital micromirror devices: principles and applications in imaging.

PubMed

Bansal, Vivek; Saggau, Peter

2013-05-01

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

DMD-based quantitative phase microscopy and optical diffraction tomography

NASA Astrophysics Data System (ADS)

Zhou, Renjie

2018-02-01

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

Nanomusical systems visualized and controlled in 4D electron microscopy.

PubMed

Baskin, J Spencer; Park, Hyun Soon; Zewail, Ahmed H

2011-05-11

Nanomusical systems, nanoharp and nanopiano, fabricated as arrays of cantilevers by focused ion beam milling of a layered Ni/Ti/Si(3)N(4) thin film, have been investigated in 4D electron microscopy. With the imaging and selective femtosecond and nanosecond control combinations, full characterization of the amplitude and phase of the resonant response of a particular cantilever relative to the optical pulse train was possible. Using a high repetition rate, low energy optical pulse train for selective, resonant excitation, coupled with pulsed and steady-state electron imaging for visualization in space and time, both the amplitude on the nanoscale and resonance of motion on the megahertz scale were resolved for these systems. Tilting of the specimen allowed in-plane and out-of-plane cantilever bending and cantilever torsional motions to be identified in stroboscopic measurements of impulsively induced free vibration. Finally, the transient, as opposed to steady state, thermostat effect was observed for the layered nanocantilevers, with a sufficiently sensitive response to demonstrate suitability for in situ use in thin-film temperature measurements requiring resolutions of <10 K and 10 μm on time scales here mechanically limited to microseconds and potentially at shorter times.

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

PubMed Central

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

2017-01-01

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

Near-Field Scanning Optical Microscopy and Raman Microscopy.

NASA Astrophysics Data System (ADS)

Harootunian, Alec Tate

1987-09-01

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

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

PubMed

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

2015-05-01

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

High-Throughput Accurate Single-Cell Screening of Euglena gracilis with Fluorescence-Assisted Optofluidic Time-Stretch Microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Ito, Takuro; Jiang, Yiyue; Ozeki, Yasuyuki; Goda, Keisuke

2016-01-01

The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel) within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a method that combines the strengths of microfluidic cell focusing, optical time-stretch microscopy, and fluorescence detection used in conventional flow cytometry. Specifically, our fluorescence-assisted optofluidic time-stretch microscope consists of an optical time-stretch microscope and a fluorescence analyzer on top of a hydrodynamically focusing microfluidic device and can detect fluorescence from every E. gracilis cell in a population and simultaneously obtain its image with a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary) and nitrogen-deficient (lipid-accumulated) E. gracilis cells with a low false positive rate of 1.0%. This method holds promise for evaluating cultivation techniques and selective breeding for microalgae-based biofuel production.

Dye-enhanced reflectance and fluorescence confocal microscopy as an optical pathology tool

NASA Astrophysics Data System (ADS)

Yaroslavsky, Anna N.; Salomatina, Elena; Novak, John; Amat-Roldan, Ivan; Castano, Ana; Hamblin, Michael

2006-02-01

Early detection and precise excision of neoplasms are imperative requirements for successful cancer treatment. In this study we evaluated the use of dye-enhanced confocal microscopy as an optical pathology tool in the ex vivo trial with fresh thick non-melanoma skin cancer excisions and in vivo trial with B16F10 melanoma cancer in mice. For the experiments the tumors were rapidly stained using aqueous solutions of either toluidine blue or methylene blue and imaged using multimodal confocal microscope. Reflectance images were acquired at the wavelengths of 630nm and 650 nm. Fluorescence was excited at 630 nm and 650 nm. Fluorescence emission was registered in the range between 680 nm and 710 nm. The images were compared to the corresponding en face frozen H&E sections. The results of the study indicate confocal images of stained cancerous tissue closely resemble corresponding H&E sections both in vivo and in vitro. This remarkable similarity enables interpretation of confocal images in a manner similar to that of histopathology. The developed technique may provide an efficient real-time optical tool for detecting skin pathology.

Synthesis and characterization of CdTe nanostructures grown by RF magnetron sputtering method

NASA Astrophysics Data System (ADS)

Akbarnejad, Elaheh; Ghoranneviss, Mahmood; Hantehzadeh, Mohammad Reza

2017-08-01

In this paper, we synthesize Cadmium Telluride nanostructures by radio frequency (RF) magnetron sputtering system on soda lime glass at various thicknesses. The effect of CdTe nanostructures thickness on crystalline, optical and morphological properties has been studied by means of X-ray diffraction (XRD), UV-VIS-NIR spectrophotometry, field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM), respectively. The XRD parameters of CdTe nanostructures such as microstrain, dislocation density, and crystal size have been examined. From XRD analysis, it could be assumed that increasing deposition time caused the formation of the wurtzite hexagonal structure of the sputtered films. Optical properties of the grown nanostructures as a function of film thickness have been observed. All the films indicate more than 60% transmission over a wide range of wavelengths. The optical band gap values of the films have obtained in the range of 1.62-1.45 eV. The results indicate that an RF sputtering method succeeded in depositing of CdTe nanostructures with high purity and controllable physical properties, which is appropriate for photovoltaic and nuclear detector applications.

High-speed polarized light microscopy for in situ, dynamic measurement of birefringence properties

NASA Astrophysics Data System (ADS)

Wu, Xianyu; Pankow, Mark; Shadow Huang, Hsiao-Ying; Peters, Kara

2018-01-01

A high-speed, quantitative polarized light microscopy (QPLM) instrument has been developed to monitor the optical slow axis spatial realignment during controlled medium to high strain rate experiments at acquisition rates up to 10 kHz. This high-speed QPLM instrument is implemented within a modified drop tower and demonstrated using polycarbonate specimens. By utilizing a rotating quarter wave plate and a high-speed camera, the minimum acquisition time to generate an alignment map of a birefringent specimen is 6.1 ms. A sequential analysis method allows the QPLM instrument to generate QPLM data at the high-speed camera imaging frequency 10 kHz. The obtained QPLM data is processed using a vector correlation technique to detect anomalous optical axis realignment and retardation changes throughout the loading event. The detected anomalous optical axis realignment is shown to be associated with crack initiation, propagation, and specimen failure in a dynamically loaded polycarbonate specimen. The work provides a foundation for detecting damage in biological tissues through local collagen fiber realignment and fracture during dynamic loading.

Fluorescence microscopy for the characterization of structural integrity

NASA Technical Reports Server (NTRS)

Street, Kenneth W.; Leonhardt, Todd A.

1991-01-01

The absorption characteristics of light and the optical technique of fluorescence microscopy for enhancing metallographic interpretation are presented. Characterization of thermally sprayed coatings by optical microscopy suffers because of the tendency for misidentification of the microstructure produced by metallographic preparation. Gray scale, in bright field microscopy, is frequently the only means of differentiating the actual structural details of porosity, cracking, and debonding of coatings. Fluorescence microscopy is a technique that helps to distinguish the artifacts of metallographic preparation (pullout, cracking, debonding) from the microstructure of the specimen by color contrasting structural differences. Alternative instrumentation and the use of other dye systems are also discussed. The combination of epoxy vacuum infiltration with fluorescence microscopy to verify microstructural defects is an effective means to characterize advanced materials and to assess structural integrity.

Tutorial on photoacoustic tomography

PubMed Central

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

2016-01-01

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

Quantitative X-ray Differential Interference Contrast Microscopy

NASA Astrophysics Data System (ADS)

Nakamura, Takashi

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

Diffraction phase microscopy realized with an automatic digital pinhole

NASA Astrophysics Data System (ADS)

Zheng, Cheng; Zhou, Renjie; Kuang, Cuifang; Zhao, Guangyuan; Zhang, Zhimin; Liu, Xu

2017-12-01

We report a novel approach to diffraction phase microscopy (DPM) with automatic pinhole alignment. The pinhole, which serves as a spatial low-pass filter to generate a uniform reference beam, is made out of a liquid crystal display (LCD) device that allows for electrical control. We have made DPM more accessible to users, while maintaining high phase measurement sensitivity and accuracy, through exploring low cost optical components and replacing the tedious pinhole alignment process with an automatic pinhole optical alignment procedure. Due to its flexibility in modifying the size and shape, this LCD device serves as a universal filter, requiring no future replacement. Moreover, a graphic user interface for real-time phase imaging has been also developed by using a USB CMOS camera. Experimental results of height maps of beads sample and live red blood cells (RBCs) dynamics are also presented, making this system ready for broad adaption to biological imaging and material metrology.

Widefield fluorescence microscopy with sensor-based conjugate adaptive optics using oblique back illumination

PubMed Central

Li, Jiang; Bifano, Thomas G.; Mertz, Jerome

2016-01-01

Abstract. We describe a wavefront sensor strategy for the implementation of adaptive optics (AO) in microscope applications involving thick, scattering media. The strategy is based on the exploitation of multiple scattering to provide oblique back illumination of the wavefront-sensor focal plane, enabling a simple and direct measurement of the flux-density tilt angles caused by aberrations at this plane. Advantages of the sensor are that it provides a large measurement field of view (FOV) while requiring no guide star, making it particularly adapted to a type of AO called conjugate AO, which provides a large correction FOV in cases when sample-induced aberrations arise from a single dominant plane (e.g., the sample surface). We apply conjugate AO here to widefield (i.e., nonscanning) fluorescence microscopy for the first time and demonstrate dynamic wavefront correction in a closed-loop implementation. PMID:27653793

Mapping carrier diffusion in single silicon core-shell nanowires with ultrafast optical microscopy.

PubMed

Seo, M A; Yoo, J; Dayeh, S A; Picraux, S T; Taylor, A J; Prasankumar, R P

2012-12-12

Recent success in the fabrication of axial and radial core-shell heterostructures, composed of one or more layers with different properties, on semiconductor nanowires (NWs) has enabled greater control of NW-based device operation for various applications. (1-3) However, further progress toward significant performance enhancements in a given application is hindered by the limited knowledge of carrier dynamics in these structures. In particular, the strong influence of interfaces between different layers in NWs on transport makes it especially important to understand carrier dynamics in these quasi-one-dimensional systems. Here, we use ultrafast optical microscopy (4) to directly examine carrier relaxation and diffusion in single silicon core-only and Si/SiO(2) core-shell NWs with high temporal and spatial resolution in a noncontact manner. This enables us to reveal strong coherent phonon oscillations and experimentally map electron and hole diffusion currents in individual semiconductor NWs for the first time.

Towards controlling molecular motions in fluorescence microscopy and optical trapping: a spatiotemporal approach

PubMed Central

Kumar De, Arijit; Goswami, Debabrata

2013-01-01

This account reviews some recent studies pursued in our group on several control experiments with important applications in (one-photon) confocal and two-photon fluorescence laser-scanning microscopy and optical trapping with laser tweezers. We explore the simultaneous control of internal and external (i.e. centre-of-mass motion) degrees of freedom, which require the coupling of various control parameters to result in the spatiotemporal control. Of particular interest to us is the implementation of such control schemes in living systems. A live cell is a system of a large number of different molecules which combine and interact to generate complex structures and functions. These combinations and interactions of molecules need to be choreographed perfectly in time and space to achieve intended intra-cellular functions. Spatiotemporal control promises to be a versatile tool for dynamical control of spatially manipulated bio-molecules. PMID:23814326

Imaging galectin-3 dependent endocytosis with lattice light-sheet microscopy

NASA Astrophysics Data System (ADS)

Baek, Jongho; Lou, Jieqiong; Coelho, Simao; Lim, Yean Jin; Seidlitz, Silvia; Nicovich, Philip R.; Wunder, Christian; Johannes, Ludger; Gaus, Katharina

2017-04-01

Lattice light-sheet (LLS) microscopy provides ultrathin light sheets of a two-dimensional optical lattice that allows us imaging three-dimensional (3D) objects for hundreds of time points at sub-second intervals and at or below the diffraction limit. Galectin-3 (Gal3), a carbohydrate-binding protein, triggers glycosphingolipid (GSL)-dependent biogenesis of morphologically distinct endocytic vesicles that are cargo specific and clathrin independent. In this study, we apply LLS microscopy to study the dynamics of Gal3 dependent endocytosis in live T cells. This will allow us to observe Gal3-mediated endocytosis at high temporal and excellent 3D spatial resolution, which may shed light on our understanding of the mechanism and physiological function of Gal3-induced endocytosis.

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

PubMed

Haustein, Elke; Schwille, Petra

2007-09-01

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

Trends in fluorescence imaging and related techniques to unravel biological information

PubMed Central

Haustein, Elke; Schwille, Petra

2007-01-01

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

Optical phase nanoscopy in red blood cells using low-coherence spectroscopy.

PubMed

Shock, Itay; Barbul, Alexander; Girshovitz, Pinhas; Nevo, Uri; Korenstein, Rafi; Shaked, Natan T

2012-10-01

We propose a low-coherence spectral-domain phase microscopy (SDPM) system for accurate quantitative phase measurements in red blood cells (RBCs) for the prognosis and monitoring of disease conditions that affect the visco-elastic properties of RBCs. Using the system, we performed time-recordings of cell membrane fluctuations, and compared the nano-scale fluctuation dynamics of healthy and glutaraldehyde-treated RBCs. Glutaraldehyde-treated RBCs possess lower amplitudes of fluctuations, reflecting an increased membrane stiffness. To demonstrate the ability of our system to measure fluctuations of lower amplitudes than those measured by the commonly used holographic phase microscopy techniques, we also constructed wide-field digital interferometry (WFDI) system and compared the performances of both systems. Due to its common-path geometry, the optical-path-delay stability of SDPM was found to be less than 0.3 nm in liquid environment, at least three times better than WFDI under the same conditions. In addition, due to the compactness of SDPM and its inexpensive and robust design, the system possesses a high potential for clinical applications.

Impact of nanostructured thin ZnO film in ultraviolet protection

PubMed Central

Sasani Ghamsari, Morteza; Alamdari, Sanaz; Han, Wooje; Park, Hyung-Ho

2017-01-01

Nanoscale ZnO is one of the best choices for ultraviolet (UV) protection, not only because of its antimicrobial properties but also due to its potential application for UV preservation. However, the behavior of nanostructured thin ZnO films and long-term effects of UV-radiation exposure have not been studied yet. In this study, we investigated the UV-protection ability of sol gel-derived thin ZnO films after different exposure times. Scanning electron microscopy, atomic force microscopy, and UV-visible optical spectroscopy were carried out to study the structure and optical properties of the ZnO films as a function of the UV-irradiation time. The results obtained showed that the prepared thin ZnO films were somewhat transparent under the visible wavelength region and protective against UV radiation. The UV-protection factor was 50+ for the prepared samples, indicating that they were excellent UV protectors. The deposited thin ZnO films demonstrated promising antibacterial potential and significant light absorbance in the UV range. The experimental results suggest that the synthesized samples have potential for applications in the health care field. PMID:28096668

Impact of nanostructured thin ZnO film in ultraviolet protection.

PubMed

Sasani Ghamsari, Morteza; Alamdari, Sanaz; Han, Wooje; Park, Hyung-Ho

2017-01-01

Nanoscale ZnO is one of the best choices for ultraviolet (UV) protection, not only because of its antimicrobial properties but also due to its potential application for UV preservation. However, the behavior of nanostructured thin ZnO films and long-term effects of UV-radiation exposure have not been studied yet. In this study, we investigated the UV-protection ability of sol gel-derived thin ZnO films after different exposure times. Scanning electron microscopy, atomic force microscopy, and UV-visible optical spectroscopy were carried out to study the structure and optical properties of the ZnO films as a function of the UV-irradiation time. The results obtained showed that the prepared thin ZnO films were somewhat transparent under the visible wavelength region and protective against UV radiation. The UV-protection factor was 50+ for the prepared samples, indicating that they were excellent UV protectors. The deposited thin ZnO films demonstrated promising antibacterial potential and significant light absorbance in the UV range. The experimental results suggest that the synthesized samples have potential for applications in the health care field.

Imaging cellular and subcellular structure of human brain tissue using micro computed tomography

NASA Astrophysics Data System (ADS)

Khimchenko, Anna; Bikis, Christos; Schweighauser, Gabriel; Hench, Jürgen; Joita-Pacureanu, Alexandra-Teodora; Thalmann, Peter; Deyhle, Hans; Osmani, Bekim; Chicherova, Natalia; Hieber, Simone E.; Cloetens, Peter; Müller-Gerbl, Magdalena; Schulz, Georg; Müller, Bert

2017-09-01

Brain tissues have been an attractive subject for investigations in neuropathology, neuroscience, and neurobiol- ogy. Nevertheless, existing imaging methodologies have intrinsic limitations in three-dimensional (3D) label-free visualisation of extended tissue samples down to (sub)cellular level. For a long time, these morphological features were visualised by electron or light microscopies. In addition to being time-consuming, microscopic investigation includes specimen fixation, embedding, sectioning, staining, and imaging with the associated artefacts. More- over, optical microscopy remains hampered by a fundamental limit in the spatial resolution that is imposed by the diffraction of visible light wavefront. In contrast, various tomography approaches do not require a complex specimen preparation and can now reach a true (sub)cellular resolution. Even laboratory-based micro computed tomography in the absorption-contrast mode of formalin-fixed paraffin-embedded (FFPE) human cerebellum yields an image contrast comparable to conventional histological sections. Data of a superior image quality was obtained by means of synchrotron radiation-based single-distance X-ray phase-contrast tomography enabling the visualisation of non-stained Purkinje cells down to the subcellular level and automated cell counting. The question arises, whether the data quality of the hard X-ray tomography can be superior to optical microscopy. Herein, we discuss the label-free investigation of the human brain ultramorphology be means of synchrotron radiation-based hard X-ray magnified phase-contrast in-line tomography at the nano-imaging beamline ID16A (ESRF, Grenoble, France). As an example, we present images of FFPE human cerebellum block. Hard X-ray tomography can provide detailed information on human tissues in health and disease with a spatial resolution below the optical limit, improving understanding of the neuro-degenerative diseases.

Hyperlens-array-implemented optical microscopy

NASA Astrophysics Data System (ADS)

Iwanaga, Masanobu

2014-08-01

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

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

PubMed

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

2014-08-25

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

Assessment of nerve ultrastructure by fibre-optic confocal microscopy.

PubMed

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

1996-01-01

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

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

PubMed

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

2014-03-01

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

Real-Space Mapping of the Chiral Near-Field Distributions in Spiral Antennas and Planar Metasurfaces.

PubMed

Schnell, M; Sarriugarte, P; Neuman, T; Khanikaev, A B; Shvets, G; Aizpurua, J; Hillenbrand, R

2016-01-13

Chiral antennas and metasurfaces can be designed to react differently to left- and right-handed circularly polarized light, which enables novel optical properties such as giant optical activity and negative refraction. Here, we demonstrate that the underlying chiral near-field distributions can be directly mapped with scattering-type scanning near-field optical microscopy employing circularly polarized illumination. We apply our technique to visualize, for the first time, the circular-polarization selective nanofocusing of infrared light in Archimedean spiral antennas, and explain this chiral optical effect by directional launching of traveling waves in analogy to antenna theory. Moreover, we near-field image single-layer rosette and asymmetric dipole-monopole metasurfaces and find negligible and strong chiral optical near-field contrast, respectively. Our technique paves the way for near-field characterization of optical chirality in metal nanostructures, which will be essential for the future development of chiral antennas and metasurfaces and their applications.

Fast Neuronal Imaging using Objective Coupled Planar Illumination Microscopy

NASA Astrophysics Data System (ADS)

Tarantino, Walter

Complex computations performed by the brain are produced by activities of neuronal populations. There is a large diversity in the functions of each individual neuron, and neuronal activities occur in the time scale of milliseconds. In order to gain a fundamental understanding of the neuronal populations, one has to measure activity of each neuron at high temporal resolution, while investigating enough neurons to encapsulate the neuronal diversity. Traditional neurotechniques such as electrophysiology and optical imaging are constrained by the number of neurons whose activities can be simultaneously measured or the speed of measuring such activities. We have developed a novel light-sheet based technique called Objective Coupled Planar Illumination (OCPI) microscopy which is capable of measuring simultaneous activities of thousands of neurons at high speeds. In this thesis I pursue the following two aims: · Improve OCPI microscopy by enhancing the spatial resolution deeper in tissue. Tissue inhomogeneity and refractive index mismatch at the surface of the tissue lead to optical aberrations. We have compensated for such aberrations by (1) miniaturizing the OCPI illumination optics, so as to enable more vertical imaging of the tissue, (2) correcting for the angular defocus caused by the refraction at the immersion fluid/tissue interface, and (3) applying adaptive optics to correct for higher order optical aberrations. The improvement in the depth at which one can image tissue will enable the measurement of activities of neuronal populations in cortical areas. · Measure the diversity in the expression pattern of VSNs responsive to sulfated steroids. Nodari et al. have identified sulfated steroids as a novel family of ligands which activate vomeronasal sensory neurons (VSNs). Due to the experimental constraints, it has not been possible to obtain a comprehensive understanding of the number, location and functional characteristics of the sulfated steroid responsive VSNs. Applying OCPI microscopy and calcium imaging to simultaneously image thousands of VSNs, we show that the sulfated steroid responsive neurons (1) have unique ligand preferences, (2) are predominantly present in the apical regions of the VNO, and (3) that the choice of expression of a receptor type is not purely stochastic.

Morphological evolution in dewetting polystyrene/polyhedral oligomeric silsesquioxane thin film bilayers.

PubMed

Paul, Rituparna; Karabiyik, Ufuk; Swift, Michael C; Hottle, John R; Esker, Alan R

2008-05-06

Morphological evolution in dewetting thin film bilayers of polystyrene (PS) and a polyhedral oligomeric silsesquioxane (POSS), trisilanolphenyl-POSS (TPP), was studied as a function of annealing temperature and annealing time. The results demonstrate unique dewetting morphologies in PS/TPP bilayers at elevated temperatures that are significantly different from those typically observed in dewetting polymer/polymer bilayers. During temperature ramp studies by optical microscopy (OM) in the reflection mode, PS/TPP bilayers form cracks with a weak optical contrast at approximately 130 degrees C. The crack formation is attributed to tensile stresses within the upper TPP layer. The weak optical contrast of the cracks observed in the bilayers for annealing temperatures below approximately 160 degrees C is consistent with the cracking and dewetting of only the upper TPP layer from the underlying PS layer. The optical contrast of the morphological features is significantly enhanced at annealing temperatures of >160 degrees C. This observation suggests dewetting of both the upper TPP and the lower PS layers that results in the exposure of the silicon substrate. Upon annealing the PS/TPP bilayers at 200 degrees C in a temperature jump experiment, the upper TPP layer undergoes instantaneous cracking as observed by OM. These cracks in the upper TPP layer serve as nucleation sites for rapid dewetting and aggregation of the TPP layer, as revealed by OM and atomic force microscopy (AFM). X-ray photoelectron spectroscopy (XPS) results indicated that dewetting of the lower PS layer ensued for annealing times >5 min and progressed up to 90 min. For annealing times >90 min, OM, AFM, and XPS results revealed complete dewetting of both the layers with the formation of TPP encapsulated PS droplets.

Optical sectioning microscopy using two-frame structured illumination and Hilbert-Huang data processing

NASA Astrophysics Data System (ADS)

Trusiak, M.; Patorski, K.; Tkaczyk, T.

2014-12-01

We propose a fast, simple and experimentally robust method for reconstructing background-rejected optically-sectioned microscopic images using two-shot structured illumination approach. Innovative data demodulation technique requires two grid-illumination images mutually phase shifted by π (half a grid period) but precise phase displacement value is not critical. Upon subtraction of the two frames the input pattern with increased grid modulation is computed. The proposed demodulation procedure comprises: (1) two-dimensional data processing based on the enhanced, fast empirical mode decomposition (EFEMD) method for the object spatial frequency selection (noise reduction and bias term removal), and (2) calculating high contrast optically-sectioned image using the two-dimensional spiral Hilbert transform (HS). The proposed algorithm effectiveness is compared with the results obtained for the same input data using conventional structured-illumination (SIM) and HiLo microscopy methods. The input data were collected for studying highly scattering tissue samples in reflectance mode. In comparison with the conventional three-frame SIM technique we need one frame less and no stringent requirement on the exact phase-shift between recorded frames is imposed. The HiLo algorithm outcome is strongly dependent on the set of parameters chosen manually by the operator (cut-off frequencies for low-pass and high-pass filtering and η parameter value for optically-sectioned image reconstruction) whereas the proposed method is parameter-free. Moreover very short processing time required to efficiently demodulate the input pattern predestines proposed method for real-time in-vivo studies. Current implementation completes full processing in 0.25s using medium class PC (Inter i7 2,1 GHz processor and 8 GB RAM). Simple modification employed to extract only first two BIMFs with fixed filter window size results in reducing the computing time to 0.11s (8 frames/s).

Chapter 7: Total internal reflection fluorescence microscopy.

PubMed

Axelrod, Daniel

2008-01-01

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

Sub-40 fs, 1060-nm Yb-fiber laser enhances penetration depth in nonlinear optical microscopy of human skin

NASA Astrophysics Data System (ADS)

Balu, Mihaela; Saytashev, Ilyas; Hou, Jue; Dantus, Marcos; Tromberg, Bruce J.

2015-12-01

Advancing the practical utility of nonlinear optical microscopy requires continued improvement in imaging depth and contrast. We evaluated second-harmonic generation (SHG) and third-harmonic generation images from ex vivo human skin and showed that a sub-40 fs, 1060-nm Yb-fiber laser can enhance SHG penetration depth by up to 80% compared to a >100 fs, 800 nm Ti:sapphire source. These results demonstrate the potential of fiber-based laser systems to address a key performance limitation related to nonlinear optical microscopy (NLOM) technology while providing a low-barrier-to-access alternative to Ti:sapphire sources that could help accelerate the movement of NLOM into clinical practice.

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

PubMed Central

Wu, Linxi

2014-01-01

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

Compensation of temporal and spatial dispersion for multiphoton acousto-optic laser-scanning microscopy

NASA Astrophysics Data System (ADS)

Iyer, Vijay; Saggau, Peter

2003-10-01

In laser-scanning microscopy, acousto-optic (AO) deflection provides a means to quickly position a laser beam to random locations throughout the field-of-view. Compared to conventional laser-scanning using galvanometer-driven mirrors, this approach increases the frame rate and signal-to-noise ratio, and reduces time spent illuminating sites of no interest. However, random-access AO scanning has not yet been combined with multi-photon microscopy, primarily because the femtosecond laser pulses employed are subject to significant amounts of both spatial and temporal dispersion upon propagation through common AO materials. Left uncompensated, spatial dispersion reduces the microscope"s spatial resolution while temporal dispersion reduces the multi-photon excitation efficacy. In previous work, we have demonstrated, 1) the efficacy of a single diffraction grating scheme which reduces the spatial dispersion at least 3-fold throughout the field-of-view, and 2) the use of a novel stacked-prism pre-chirper for compensating the temporal dispersion of a pair of AODs using a shorter mechanical path length (2-4X) than standard prism-pair arrangements. In this work, we demonstrate for the first time the use of these compensation approaches with a custom-made large-area slow-shear TeO2 AOD specifically suited for the development of a high-resolution 2-D random-access AO scanning multi-photon laser-scanning microscope (AO-MPLSM).

eduSPIM: Light Sheet Microscopy in the Museum.

PubMed

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

2016-01-01

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

Real-time assessment of breast surgical margins with fluorescence-guided microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Iftimia, Nicusor V.; Park, Jesung; Maguluri, Gopi N.; Krishnamurthy, Savitri

2017-02-01

A novel multimodal optical imaging approach for real-time assessment of surgical margins on breast cancer lumpectomy specimens is presented. Our approach is to target cancer cells using an optically silent peptide substrate containing two (NIR) fluorochromes, internally quenched, which are cleaved by highly expressed breast cancer enzymes, like urokinase-type plasminogen activator (uPA). Thus this agent becomes highly fluorescent only on the cancer area when the specimen is excited by a NIR laser beam. A fluorescence imager is used to highlight cancer-suspect margins on the surgical specimen, while high-resolution optical coherence tomography (OCT) imaging is used to visualize tissue morphology on the highlighted areas and confirm or rule out cancer presence. This technology will hopefully increase the success rate of cancer surgeries, reduce the risk of cancer recurrence and significantly reduce US healthcare costs.

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

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

PubMed

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

2018-05-21

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

Influence of interface layer on optical properties of sub-20 nm-thick TiO2 films

NASA Astrophysics Data System (ADS)

Shi, Yue-Jie; Zhang, Rong-Jun; Li, Da-Hai; Zhan, Yi-Qiang; Lu, Hong-Liang; Jiang, An-Quan; Chen, Xin; Liu, Juan; Zheng, Yu-Xiang; Wang, Song-You; Chen, Liang-Yao

2018-02-01

The sub-20 nm ultrathin titanium dioxide (TiO2) films with tunable thickness were deposited on Si substrates by atomic layer deposition (ALD). The structural and optical properties were acquired by transmission electron microscopy, atomic force microscopy and spectroscopic ellipsometry. Afterwards, a constructive and effective method of analyzing interfaces by applying two different optical models consisting of air/TiO2/Ti x Si y O2/Si and air/effective TiO2 layer/Si, respectively, was proposed to investigate the influence of interface layer (IL) on the analysis of optical constants and the determination of band gap of TiO2 ultrathin films. It was found that two factors including optical constants and changing components of the nonstoichiometric IL could contribute to the extent of the influence. Furthermore, the investigated TiO2 ultrathin films of 600 ALD cycles were selected and then annealed at the temperature range of 400-900 °C by rapid thermal annealing. Thicker IL and phase transition cause the variation of optical properties of TiO2 films after annealing and a shorter electron relaxation time reveals the strengthened electron-electron and electron-phonon interactions in the TiO2 ultrathin films at high temperature. The as-obtained results in this paper will play a role in other studies of high dielectric constants materials grown on Si substrates and in the applications of next generation metal-oxide-semiconductor devices.

Motion compensation for in vivo subcellular optical microscopy.

PubMed

Lucotte, B; Balaban, R S

2014-04-01

In this review, we focus on the impact of tissue motion on attempting to conduct subcellular resolution optical microscopy, in vivo. Our position is that tissue motion is one of the major barriers in conducting these studies along with light induced damage, optical probe loading as well as absorbing and scattering effects on the excitation point spread function and collection of emitted light. Recent developments in the speed of image acquisition have reached the limit, in most cases, where the signal from a subcellular voxel limits the speed and not the scanning rate of the microscope. Different schemes for compensating for tissue displacements due to rigid body and deformation are presented from tissue restriction, gating, adaptive gating and active tissue tracking. We argue that methods that minimally impact the natural physiological motion of the tissue are desirable because the major reason to perform in vivo studies is to evaluate normal physiological functions. Towards this goal, active tracking using the optical imaging data itself to monitor tissue displacement and either prospectively or retrospectively correct for the motion without affecting physiological processes is desirable. Critical for this development was the implementation of near real time image processing in conjunction with the control of the microscope imaging parameters. Clearly, the continuing development of methods of motion compensation as well as significant technological solutions to the other barriers to tissue subcellular optical imaging in vivo, including optical aberrations and overall signal-to-noise ratio, will make major contributions to the understanding of cell biology within the body.

Polyaniline decorated Bi2MoO6 nanosheets with effective interfacial charge transfer as photocatalysts and optical limiters.

PubMed

Zhao, Wei; Li, Cheng; Wang, Aijian; Lv, Cuncai; Zhu, Weihua; Dou, Shengping; Wang, Qian; Zhong, Qin

2017-11-01

Polyaniline (PANI)-decorated Bi 2 MoO 6 nanosheets (BMO/PANI) were prepared by a facile solvothermal method. Different characterization techniques, including X-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, Raman spectroscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, diffuse reflectance ultraviolet-visible spectroscopy, photoluminescence spectroscopy, electrochemical impedance spectroscopy, photocurrent spectroscopy, and nanosecond time-resolved emission studies, have been employed to investigate the structure, optical and electrical properties of the BMO/PANI composites. The wide absorption of the samples in the visible light region makes them suitable for nonlinear transmission and photocatalytic activity studies. The associated photocatalytic activity and optical nonlinearities for the BMO/PANI composites are shown to be dependent on the PANI loadings. The rational mechanisms responsible for deteriorating pollutants and improving optical nonlinearities were also proposed, which could be mainly attributed to the efficient interfacial charge transfer and the interfacial electronic interactions between PANI and Bi 2 MoO 6 . The photoluminescence spectroscopy, electrochemical impedance spectroscopy, and photocurrent spectroscopy studies confirmed that the interface charge separation efficiency was greatly improved by coupling Bi 2 MoO 6 with PANI. The tuning of photocatalysis and nonlinear optical behaviors with variation in the content of PANI provides an easy way to attain tunable properties, which are exceedingly required in optoelectronics applications.

Large electro-optic coefficient in single-crystal film of a novel organic salt, DASMS

NASA Astrophysics Data System (ADS)

Tan, Shida; Ahyi, Ayayi; Mishra, Alpana; Thakur, Mrinal

2001-03-01

We have synthesized a novel electro-optic material 4'-dimethylamino-4-methylstilbazolium methanesulfonate (DASMS). Large-area ( 60 mm^2), single-crystal films of DASMS with excellent optical quality have been grown for the first time by a modified shear method^1. These films have the noncentrosymmetric hydrated phase, which is electro-optically active^2. Polarized optical microscopy, X-ray diffraction and polarized UV-visible spectroscopic studies have been used to characterize the films. The single-crystal films were observed to be highly dichroic. Using field-induced birefringence measurement, the electro-optic coefficient of DASMS at 632.8 nm has been estimated to be r_11 160 pm/V, which is five times larger than the eletro-optic coefficient of LiNbO_3. For a 1.8 μm thick film, 28% intensity modulation was observed for an electric field of 4 V/μm. 1. M. Thakur and S. Meyler, Macromolecules 18, 2341 (1985); M. Thakur, Y. Shani, G. C. Chi, and K. O'Brien, Synth. Met. 28, D595 (1989). 2. E. P. Boden, P. D. Phelps, C. P. Yakymyshyn, and K. R. Stewart, US patent 5,194,584.

Leakage radiation interference microscopy.

PubMed

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

2013-09-01

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

Preparation of CuO nanoparticles by laser ablation in liquid

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

Abdulateef, Sinan A., E-mail: sinan1974@yahoo.com; MatJafri, M. Z.; Omar, A. F., E-mail: thinker-academy@yahoo.com

2016-07-06

Colloidal Cu nanoparticles (NPs) were synthesized by pulsed Nd:YAG laser ablation in acetone. Cu NPs were converted into CuO. The size and optical properties of these NPs were characterized using an UV/Vis spectrophotometer, transmission electron microscopy, and X-ray diffraction. Cu NPs were spherical, and their mean diameter in acetone was 8 nm–10 nm. Optical extinction immediately after the ablation showed surface Plasmon resonance peaks at 602 nm. The color of Cu NPs in acetone was green and stable even after a long time.

[Gene sequencing by scanning molecular exciton microscopy]. Progress report, October 1, 1990--September 30, 1991

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

Not Available

1991-12-31

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

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

NASA Astrophysics Data System (ADS)

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

2009-11-01

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

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

PubMed

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

2014-12-11

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

Aberration control in 4Pi nanoscopy: definitions, properties, and applications (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hao, Xiang; Allgeyer, Edward S.; Velasco, Mary Grace M.; Booth, Martin J.; Bewersdorf, Joerg

2016-03-01

The development of fluorescence microscopy, which allows live-cell imaging with high labeling specificity, has made the visualization of cellular architecture routine. However, for centuries, the spatial resolution of optical microscopy was fundamentally limited by diffraction. The past two decades have seen a revolution in far-field optical nanoscopy (or "super-resolution" microscopy). The best 3D resolution is achieved by optical nanoscopes like the isoSTED or the iPALM/4Pi-SMS, which utilize two opposing objective lenses in a coherent manner. These system are, however, also more complex and the required interference conditions demand precise aberration control. Our research involves developing novel adaptive optics techniques that enable high spatial and temporal resolution imaging for biological applications. In this talk, we will discuss how adaptive optics can enhance dual-objective lens nanoscopes. We will demonstrate how adaptive optics devices provide unprecedented freedom to manipulate the light field in isoSTED nanoscopy, allow to realize automatic beam alignment, suppress the inherent side-lobes of the point-spread function, and dynamically compensate for sample-induced aberrations. We will present both the theoretical groundwork and the experimental confirmations.

Virtual k -Space Modulation Optical Microscopy

NASA Astrophysics Data System (ADS)

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

2016-07-01

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

Understanding the optics to aid microscopy image segmentation.

PubMed

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

2010-01-01

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

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

PubMed Central

Wang, Le; Xu, Xiaoji G.

2015-01-01

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

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

PubMed

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

2016-09-01

Optoacoustic (OA) microscopy using an all-optical system based on the probe beam deflection technique (PBDT) for detection of laser-induced acoustic signals was investigated as an alternative to conventional piezoelectric transducers. PBDT provides a number of advantages for OA microscopy including (i) efficient coupling of laser excitation energy to the samples being imaged through the probing laser beam, (ii) undistorted coupling of acoustic waves to the detector without the need for separation of the optical and acoustic paths, (iii) high sensitivity and (iv) ultrawide bandwidth. Because of the unimpeded optical path in PBDT, diffraction-limited lateral resolution can be readily achieved. The sensitivity of the current PBDT sensor of 22 μV/Pa and its noise equivalent pressure (NEP) of 11.4 Pa are comparable with these parameters of the optical micro-ring resonator and commercial piezoelectric ultrasonic transducers. Benefits of the present prototype OA microscope were demonstrated by successfully resolving micron-size details in histological sections of cardiac muscle.

Light Sheet Fluorescence Microscopy (LSFM)

PubMed Central

Adams, Michael W.; Loftus, Andrew F.; Dunn, Sarah E.; Joens, Matthew S.; Fitzpatrick, James A.J.

2015-01-01

The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light Sheet Fluorescent Microscopy (LSFM), a century old idea (Siedentopf and Zsigmondy, 1902) made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light sheet based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements. PMID:25559221

Planar Diffractive Lenses: Fundamentals, Functionalities, and Applications.

PubMed

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

2018-06-01

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

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

NASA Astrophysics Data System (ADS)

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

2016-03-01

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

Quantitative photoacoustic microscopy of optical absorption coefficients from acoustic spectra in the optical diffusive regime

NASA Astrophysics Data System (ADS)

Guo, Zijian; Favazza, Christopher; Garcia-Uribe, Alejandro; Wang, Lihong V.

2012-06-01

Photoacoustic (PA) microscopy (PAM) can image optical absorption contrast with ultrasonic spatial resolution in the optical diffusive regime. Conventionally, accurate quantification in PAM requires knowledge of the optical fluence attenuation, acoustic pressure attenuation, and detection bandwidth. We circumvent this requirement by quantifying the optical absorption coefficients from the acoustic spectra of PA signals acquired at multiple optical wavelengths. With the acoustic spectral method, the absorption coefficients of an oxygenated bovine blood phantom at 560, 565, 570, and 575 nm were quantified with errors of <3%. We also quantified the total hemoglobin concentration and hemoglobin oxygen saturation in a live mouse. Compared with the conventional amplitude method, the acoustic spectral method provides greater quantification accuracy in the optical diffusive regime. The limitations of the acoustic spectral method was also discussed.

Quantitative photoacoustic microscopy of optical absorption coefficients from acoustic spectra in the optical diffusive regime

PubMed Central

Guo, Zijian; Favazza, Christopher; Garcia-Uribe, Alejandro

2012-01-01

Abstract. Photoacoustic (PA) microscopy (PAM) can image optical absorption contrast with ultrasonic spatial resolution in the optical diffusive regime. Conventionally, accurate quantification in PAM requires knowledge of the optical fluence attenuation, acoustic pressure attenuation, and detection bandwidth. We circumvent this requirement by quantifying the optical absorption coefficients from the acoustic spectra of PA signals acquired at multiple optical wavelengths. With the acoustic spectral method, the absorption coefficients of an oxygenated bovine blood phantom at 560, 565, 570, and 575 nm were quantified with errors of <3%. We also quantified the total hemoglobin concentration and hemoglobin oxygen saturation in a live mouse. Compared with the conventional amplitude method, the acoustic spectral method provides greater quantification accuracy in the optical diffusive regime. The limitations of the acoustic spectral method was also discussed. PMID:22734767

Quantitative photoacoustic microscopy of optical absorption coefficients from acoustic spectra in the optical diffusive regime.

PubMed

Guo, Zijian; Favazza, Christopher; Garcia-Uribe, Alejandro; Wang, Lihong V

2012-06-01

Photoacoustic (PA) microscopy (PAM) can image optical absorption contrast with ultrasonic spatial resolution in the optical diffusive regime. Conventionally, accurate quantification in PAM requires knowledge of the optical fluence attenuation, acoustic pressure attenuation, and detection bandwidth. We circumvent this requirement by quantifying the optical absorption coefficients from the acoustic spectra of PA signals acquired at multiple optical wavelengths. With the acoustic spectral method, the absorption coefficients of an oxygenated bovine blood phantom at 560, 565, 570, and 575 nm were quantified with errors of <3%. We also quantified the total hemoglobin concentration and hemoglobin oxygen saturation in a live mouse. Compared with the conventional amplitude method, the acoustic spectral method provides greater quantification accuracy in the optical diffusive regime. The limitations of the acoustic spectral method was also discussed.

CONFERENCE NOTE: European Optical Society, Topical Meeting Optical Metrology and Nanotechnology, Engelberg, Switzerland, 27 30 March 1994

NASA Astrophysics Data System (ADS)

1993-01-01

This meeting, organized by the Paul Scherrer Institute's Department of Applied Solid State Physics, will be held from 27 30 March 1994 at the Hotel Regina-Titlis, Engelberg, Switzerland. The aim is to bring together scientists from two important fields of current research and increasing industrial relevance. Optical metrology is a traditional discipline of applied optics which reached the nanometre scale a long time ago. Nanotechnology is setting new limits and represents a major challenge to metrology, as well as offering new opportunities to optics. The meeting is intended to help define a common future for optical metrology and nanotechnology. Topics to be covered include: nanometre position control and measuring techniques ultrahigh precision interferometry scanning probe microscopy (AFM, SNOM, etc.) surface modification by scanning probe methods precision surface fabrication and characterization nanolithography micro-optics, diffractive optics components, including systems and applications subwavelength optical structures synthetic optical materials structures and technologies for X-ray optics. For further information please contact: Jens Gobrecht (Secretary), Paul Scherrer Institute, CH-5232 Villigen-PSI, Switzerland.Tel. (41)56992529; Fax (41) 5698 2635.

Light Microscopy at Maximal Precision

NASA Astrophysics Data System (ADS)

Bierbaum, Matthew; Leahy, Brian D.; Alemi, Alexander A.; Cohen, Itai; Sethna, James P.

2017-10-01

Microscopy is the workhorse of the physical and life sciences, producing crisp images of everything from atoms to cells well beyond the capabilities of the human eye. However, the analysis of these images is frequently little more accurate than manual marking. Here, we revolutionize the analysis of microscopy images, extracting all the useful information theoretically contained in a complex microscope image. Using a generic, methodological approach, we extract the information by fitting experimental images with a detailed optical model of the microscope, a method we call parameter extraction from reconstructing images (PERI). As a proof of principle, we demonstrate this approach with a confocal image of colloidal spheres, improving measurements of particle positions and radii by 10-100 times over current methods and attaining the maximum possible accuracy. With this unprecedented accuracy, we measure nanometer-scale colloidal interactions in dense suspensions solely with light microscopy, a previously impossible feat. Our approach is generic and applicable to imaging methods from brightfield to electron microscopy, where we expect accuracies of 1 nm and 0.1 pm, respectively.

Ultra-high resolution coded wavefront sensor.

PubMed

Wang, Congli; Dun, Xiong; Fu, Qiang; Heidrich, Wolfgang

2017-06-12

Wavefront sensors and more general phase retrieval methods have recently attracted a lot of attention in a host of application domains, ranging from astronomy to scientific imaging and microscopy. In this paper, we introduce a new class of sensor, the Coded Wavefront Sensor, which provides high spatio-temporal resolution using a simple masked sensor under white light illumination. Specifically, we demonstrate megapixel spatial resolution and phase accuracy better than 0.1 wavelengths at reconstruction rates of 50 Hz or more, thus opening up many new applications from high-resolution adaptive optics to real-time phase retrieval in microscopy.

Synthesis and Characteristics of ZnS Nanospheres for Heterojunction Photovoltaic Device

NASA Astrophysics Data System (ADS)

Chou, Sheng-Hung; Hsiao, Yu-Jen; Fang, Te-Hua; Chou, Po-Hsun

2015-06-01

The synthesis of ZnS nanospheres produced using the microwave hydrothermal method was studied. The microstructure and surface and optical properties of ZnS nanospheres on glass were characterized using scanning electron microscopy, high-resolution transmission electron microscopy, x-ray diffraction, and ultraviolet-visible spectroscopy. The influence of deposition time on the transmission and photovoltaic performance was determined. The power conversion efficiency of an Al-doped ZnO/ZnS nanosphere/textured p-Si device improved from 0.93 to 1.77% when the thickness of the ZnS nanostructured film was changed from 75 to 150 nm.

Attosecond electron pulses for 4D diffraction and microscopy

PubMed Central

Baum, Peter; Zewail, Ahmed H.

2007-01-01

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

Localization microscopy of DNA in situ using Vybrant{sup ®} DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution

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

Żurek-Biesiada, Dominika; Szczurek, Aleksander T.; Prakash, Kirti

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant{sup ®} DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei ofmore » fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10{sup 6} signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100 nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy. - Highlights: • Super-resolution imaging of nuclear DNA with Vybrant Violet and blue excitation. • 90nm resolution images of DNA structures in optically thick eukaryotic nuclei. • Enhanced resolution confirms the existence of DNA-free regions inside the nucleus. • Optimized imaging conditions enable multicolor super-resolution imaging.« less

Super-resolution optical microscopy study of telomere structure.

PubMed

Phipps, Mary Lisa; Goodwin, Peter M; Martinez, Jennifer S; Goodwin, Edwin H

2016-09-01

Chromosome ends are shielded from exonucleolytic attack and inappropriate end-joining by terminal structures called telomeres; these structures are potential targets for anticancer drugs. Telomeres are composed of a simple DNA sequence (5?-TTAGGG-3? in humans) repeated more than a thousand times, a short 3? single-stranded overhang, and numerous proteins. Electron microscopy has shown that the 3? overhang pairs with the complementary strand at an internal site creating a small displacement loop and a large double-stranded “t-loop.” Our goal is to determine whether all telomeres adopt the t-loop configuration, or whether there are two or more distinct configurations. Progress in optimizing super-resolution (SR) microscopy for this ongoing investigation is reported here. Results suggest that under certain conditions sample preparation procedures may disrupt chromatin by causing loss of nucleosomes. This finding may limit the use of SR microscopy in telomere studies.

Fabrication and optical characterization of imaging fiber-based nanoarrays.

PubMed

Tam, Jenny M; Song, Linan; Walt, David R

2005-09-15

In this paper, we present a technique for fabricating arrays containing a density at least 90 times higher than previously published. Specifically, we discuss the fabrication of two imaging fiber-based nanoarrays, one with 700nm features, another with 300nm features. With arrays containing up to 4.5x10(6) array elements/mm(2), these nanoarrays have an ultra-high packing density. A straightforward etching protocol is used to create nanowells into which beads can be deposited. These beads comprise the sensing elements of the nanoarray. Deposition of the nanobeads into the nanowells using two techniques is described. The surface characteristics of the etched arrays are examined with atomic force microscopy and scanning electron microscopy. Fluorescence microscopy was used to observe the arrays. The 300nm array features and the 500nm center-to-center distance approach the minimum feature sizes viewable using conventional light microscopy.

Super-resolution optical microscopy study of telomere structure

NASA Astrophysics Data System (ADS)

Phipps, Mary Lisa; Goodwin, Peter M.; Martinez, Jennifer S.; Goodwin, Edwin H.

2016-09-01

Chromosome ends are shielded from exonucleolytic attack and inappropriate end-joining by terminal structures called telomeres; these structures are potential targets for anticancer drugs. Telomeres are composed of a simple DNA sequence (5‧-TTAGGG-3‧ in humans) repeated more than a thousand times, a short 3‧ single-stranded overhang, and numerous proteins. Electron microscopy has shown that the 3‧ overhang pairs with the complementary strand at an internal site creating a small displacement loop and a large double-stranded "t-loop." Our goal is to determine whether all telomeres adopt the t-loop configuration, or whether there are two or more distinct configurations. Progress in optimizing super-resolution (SR) microscopy for this ongoing investigation is reported here. Results suggest that under certain conditions sample preparation procedures may disrupt chromatin by causing loss of nucleosomes. This finding may limit the use of SR microscopy in telomere studies.

Effect of T6 heat treatment on the microstructural and mechanical properties of Al-Si-Cu-Mg alloys

NASA Astrophysics Data System (ADS)

Patel, Dhruv; Davda, Chintan; Solanki, P. S.; Keshvani, M. J.

2016-05-01

In this communication, it is aimed to optimize the conditions for T6 heat treatment of permanent die cast Al-Si-Cu-Mg alloys. Various solutionizing temperatures, aging treatments and soaking times were used to improve / modify the mechanical properties of presently studied alloys. Formation mechanism of the particles was understood by carrying out optical microscopy and energy dispersive X-ray (EDX) spectroscopy measurements. Spherical particles of alloys were studied for their microstructural properties using scanning electron microscopy (SEM). Microhardness test was performed to investigate their mechanical properties. Dependence of cluster formation and microhardness of the alloys on the adequate solutionizing temperature, aging treatment and soaking time has been discussed in detail.

Laser beam shaping for biomedical microscopy techniques

NASA Astrophysics Data System (ADS)

Laskin, Alexander; Kaiser, Peter; Laskin, Vadim; Ostrun, Aleksei

2016-04-01

Uniform illumination of a working field is very important in optical systems of confocal microscopy and various implementations of fluorescence microscopy like TIR, SSIM, STORM, PALM to enhance performance of these laser-based research techniques. Widely used TEM00 laser sources are characterized by essentially non-uniform Gaussian intensity profile which leads usually to non-uniform intensity distribution in a microscope working field or in a field of microlenses array of a confocal microscope optical system, this non-uniform illumination results in instability of measuring procedure and reducing precision of quantitative measurements. Therefore transformation of typical Gaussian distribution of a TEM00 laser to flat-top (top hat) profile is an actual technical task, it is solved by applying beam shaping optics. Due to high demands to optical image quality the mentioned techniques have specific requirements to a uniform laser beam: flatness of phase front and extended depth of field, - from this point of view the microscopy techniques are similar to holography and interferometry. There are different refractive and diffractive beam shaping approaches used in laser industrial and scientific applications, but only few of them are capable to fulfil the optimum conditions for beam quality required in discussed microscopy techniques. We suggest applying refractive field mapping beam shapers πShaper, which operational principle presumes almost lossless transformation of Gaussian to flat-top beam with flatness of output wavefront, conserving of beam consistency, providing collimated low divergent output beam, high transmittance, extended depth of field, negligible wave aberration, and achromatic design provides capability to work with several lasers with different wavelengths simultaneously. The main function of a beam shaper is transformation of laser intensity profile, further beam transformation to provide optimum for a particular technique spot size and shape has to be realized by an imaging optical system which can include microscope objectives and tube lenses. This paper will describe design basics of refractive beam shapers and optical layouts of their applying in microscopy systems. Examples of real implementations and experimental results will be presented as well.

Effect of growth time on the structure, morphology and optical properties of hydrothermally synthesized TiO2 nanorod thin films

NASA Astrophysics Data System (ADS)

Mohapatra, A. K.; Nayak, J.

2018-05-01

Titanium dioxide (TiO2) nanorod thin films were deposited on fluorine doped tin oxide coated glass substrates by a single step rapid hydrothermal process. The concentration of the precursor, the temperature of the reaction mixture were optimized in order to enhance the rate of deposition. Unlike the previously reported hydrothermal treatment for 24 - 48 h, the deposition of well aligned titanium dioxide nanorods was achieved in a short time such as 3 - 8 h. The crystal structure of the films were investigated by X-rays diffraction. The morphology of the nanorod films were studied with scanning electron microscopy. The optical properties were studied by photoluminescence spectroscopy.

Hemodynamic flow visualization of early embryonic great vessels using μPIV.

PubMed

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

2015-01-01

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

Growth and nonlinear optical characterization of organic single crystal films

NASA Astrophysics Data System (ADS)

Zhou, Ligui

1997-12-01

Organic single crystal films are important for various future applications in photonics and integrated optics. The conventional method for inorganic crystal growth is not suitable for organic materials, and the high temperature melting method is not good for most organic materials due to decomposition problems. We developed a new method-modified shear method-to grow large area organic single crystal thin films which have exceptional nonlinear optical properties and high quality surfaces. Several organic materials (NPP, PNP and DAST) were synthesized and purified before the thin film crystal growth. Organic single crystal thin films were grown from saturated organic solutions using modified shear method. The area of single crystal films were about 1.5 cm2 for PNP, 1 cm2 for NPP and 5 mm2 for DAST. The thickness of the thin films which could be controlled by the applied pressure ranged from 1μm to 10 μm. The single crystal thin films of organic materials were characterized by polarized microscopy, x-ray diffraction, polarized UV-Visible and polarized micro-FTIR spectroscopy. Polarized microscopy showed uniform birefringence and complete extinction with the rotation of the single crystal thin films under crossed- polarization, which indicated high quality single crystals with no scattering. The surface orientation of single crystal thin films was characterized by x-ray diffraction. The molecular orientation within the crystal was further studied by the polarized UV-Visible and Polarized micro-FTIR techniques combined with the x-ray and polarized microscopy results. A Nd:YAG laser with 35 picosecond pulses at 1064nm wavelength was employed to perform the nonlinear optical characterization of the organic single crystal thin films. Two measurement techniques were used to study the crystal films: second harmonic generation (SHG) and electro-optic (EO) effect. SHG results showed that the nonlinear optical coefficient of NPP was 18 times that of LiNbO3, a standard inorganic crystal material, and the nonlinear optical coefficient of PNP was 11 times that of LiNbO3. Electro-optic measurements showed that r11 = 65 pm/V for NPP and r12 = 350 pm/V for DAST. EO modulation effect was also observed using Fabry-Perot interferometry. Waveguide devices are very important for integrated optics. But the fabrication of waveguide devices on the organic single crystal thin films was difficult due to the solubility of the film in common organic solvents. A modified photolithographic technique was employed to make channel waveguides and poly(vinyl alcohol) (PVA) was used as a protective layer in the fabrication of the waveguides. Waveguides with dimensions about 7/mum x 1μm x 1mm were obtained.

Photoacoustic microscopy of human teeth

NASA Astrophysics Data System (ADS)

Rao, Bin; Cai, Xin; Favazza, Christopher; Yao, Junjie; Li, Li; Duong, Steven; Liaw, Lih-Huei; Holtzman, Jennifer; Wilder-Smith, Petra; Wang, Lihong V.

2011-03-01

Photoacoustic microscopy (PAM) utilizes short laser pulses to deposit energy into light absorbers and sensitively detects the ultrasonic waves the absorbers generate in response. PAM directly renders a three-dimensional spatial distribution of sub-surface optical absorbers. Unlike other optical imaging technologies, PAM features label-free optical absorption contrast and excellent imaging depths. Standard dental imaging instruments are limited to X-ray and CCD cameras. Subsurface optical dental imaging is difficult due to the highly-scattering enamel and dentin tissue. Thus, very few imaging methods can detect dental decay or diagnose dental pulp, which is the innermost part of the tooth, containing the nerves, blood vessels, and other cells. Here, we conducted a feasibility study on imaging dental decay and dental pulp with PAM. Our results showed that PAM is sensitive to the color change associated with dental decay. Although the relative PA signal distribution may be affected by surface contours and subsurface reflections from deeper dental tissue, monitoring changes in the PA signals (at the same site) over time is necessary to identify the progress of dental decay. Our results also showed that deep-imaging, near-infrared (NIR) PAM can sensitively image blood in the dental pulp of an in vitro tooth. In conclusion, PAM is a promising tool for imaging both dental decay and dental pulp.

Characterization of Si3N4/SiO2 optical channel waveguides by photon scanning tunneling microscopy

NASA Technical Reports Server (NTRS)

Wang, Yan; Chudgar, Mona H.; Jackson, Howard E.; Miller, Jeffrey S.; De Brabander, Gregory N.; Boyd, Joseph T.

1993-01-01

Photon scanning tunneling microscopy (PSTM) is used to characterize Si3N4/Si02 optical channel waveguides being used for integrated optical-micromechanical sensors. PSTM utilizes an optical fiber tapered to a fine point which is piezoelectrically positioned to measure the decay of the evanescent field intensity associated with the waveguide propagating mode. Evanescent field decays are recorded for both ridge channel waveguides and planar waveguide regions. Values for the local effective refractive index are calculated from the data for both polarizations and compared to model calculations.

DNA-carbon nano onion aggregate: triangle, hexagon, six-petal flower to dead-end network

NASA Astrophysics Data System (ADS)

Babar, Dipak Gorakh; Pakhira, Bholanath; Sarkar, Sabyasachi

2017-08-01

The interaction between calf-thymus (CT) dsDNA and water soluble carbon nano onion (wsCNO) in water follows denaturation of dsDNA (double stranded) to ssDNA (single stranded) as monitored by optical spectroscopy. The ssDNA concomitantly wraps the spiky surface of wsCNO to create triangular aggregate as the building block as observed by time-dependent SEM images. These triangles further aggregate leading to six-petal flower arrangement via hexagon and finally reach a dead end network as imaged by SEM and optical fluorescence microscopy. The dead-end network aggregate lost the intrinsic optical property of DNA suggesting complete loss of its activity.

Live imaging using adaptive optics with fluorescent protein guide-stars

PubMed Central

Tao, Xiaodong; Crest, Justin; Kotadia, Shaila; Azucena, Oscar; Chen, Diana C.; Sullivan, William; Kubby, Joel

2012-01-01

Spatially and temporally dependent optical aberrations induced by the inhomogeneous refractive index of live samples limit the resolution of live dynamic imaging. We introduce an adaptive optical microscope with a direct wavefront sensing method using a Shack-Hartmann wavefront sensor and fluorescent protein guide-stars for live imaging. The results of imaging Drosophila embryos demonstrate its ability to correct aberrations and achieve near diffraction limited images of medial sections of large Drosophila embryos. GFP-polo labeled centrosomes can be observed clearly after correction but cannot be observed before correction. Four dimensional time lapse images are achieved with the correction of dynamic aberrations. These studies also demonstrate that the GFP-tagged centrosome proteins, Polo and Cnn, serve as excellent biological guide-stars for adaptive optics based microscopy. PMID:22772285

Grid-enhanced X-ray coded aperture microscopy with polycapillary optics

PubMed Central

Sowa, Katarzyna M.; Last, Arndt; Korecki, Paweł

2017-01-01

Polycapillary devices focus X-rays by means of multiple reflections of X-rays in arrays of bent glass capillaries. The size of the focal spot (typically 10–100 μm) limits the resolution of scanning, absorption and phase-contrast X-ray imaging using these devices. At the expense of a moderate resolution, polycapillary elements provide high intensity and are frequently used for X-ray micro-imaging with both synchrotrons and X-ray tubes. Recent studies have shown that the internal microstructure of such an optics can be used as a coded aperture that encodes high-resolution information about objects located inside the focal spot. However, further improvements to this variant of X-ray microscopy will require the challenging fabrication of tailored devices with a well-defined capillary microstructure. Here, we show that submicron coded aperture microscopy can be realized using a periodic grid that is placed at the output surface of a polycapillary optics. Grid-enhanced X-ray coded aperture microscopy with polycapillary optics does not rely on the specific microstructure of the optics but rather takes advantage only of its focusing properties. Hence, submicron X-ray imaging can be realized with standard polycapillary devices and existing set-ups for micro X-ray fluorescence spectroscopy. PMID:28322316

Grid-enhanced X-ray coded aperture microscopy with polycapillary optics.

PubMed

Sowa, Katarzyna M; Last, Arndt; Korecki, Paweł

2017-03-21

Polycapillary devices focus X-rays by means of multiple reflections of X-rays in arrays of bent glass capillaries. The size of the focal spot (typically 10-100 μm) limits the resolution of scanning, absorption and phase-contrast X-ray imaging using these devices. At the expense of a moderate resolution, polycapillary elements provide high intensity and are frequently used for X-ray micro-imaging with both synchrotrons and X-ray tubes. Recent studies have shown that the internal microstructure of such an optics can be used as a coded aperture that encodes high-resolution information about objects located inside the focal spot. However, further improvements to this variant of X-ray microscopy will require the challenging fabrication of tailored devices with a well-defined capillary microstructure. Here, we show that submicron coded aperture microscopy can be realized using a periodic grid that is placed at the output surface of a polycapillary optics. Grid-enhanced X-ray coded aperture microscopy with polycapillary optics does not rely on the specific microstructure of the optics but rather takes advantage only of its focusing properties. Hence, submicron X-ray imaging can be realized with standard polycapillary devices and existing set-ups for micro X-ray fluorescence spectroscopy.

Fabrication of bright and thin Zn₂SiO₄ luminescent film for electron beam excitation-assisted optical microscope.

PubMed

Furukawa, Taichi; Kanamori, Satoshi; Fukuta, Masahiro; Nawa, Yasunori; Kominami, Hiroko; Nakanishi, Yoichiro; Sugita, Atsushi; Inami, Wataru; Kawata, Yoshimasa

2015-07-13

We fabricated a bright and thin Zn₂SiO₄ luminescent film to serve as a nanometric light source for high-spatial-resolution optical microscopy based on electron beam excitation. The Zn₂SiO₄ luminescent thin film was fabricated by annealing a ZnO film on a Si₃N₄ substrate at 1000 °C in N₂. The annealed film emitted bright cathodoluminescence compared with the as-deposited film. The film is promising for nano-imaging with electron beam excitation-assisted optical microscopy. We evaluated the spatial resolution of a microscope developed using this Zn₂SiO₄ luminescent thin film. This is the first report of the investigation and application of ZnO/Si₃N₄ annealed at a high temperature (1000 °C). The fabricated Zn₂SiO₄ film is expected to enable high-frame-rate dynamic observation with ultra-high resolution using our electron beam excitation-assisted optical microscopy.

Electro-optical interfacial effects on a graphene/π-conjugated organic semiconductor hybrid system

PubMed Central

Araujo, Karolline A S; Cury, Luiz A; Matos, Matheus J S; Fernandes, Thales F D; Cançado, Luiz G

2018-01-01

The influence of graphene and retinoic acid (RA) – a π-conjugated organic semiconductor – interface on their hybrid system is investigated. The physical properties of the interface are assessed via scanning probe microscopy, optical spectroscopy (photoluminescence and Raman) and ab initio calculations. The graphene/RA interaction induces the formation of a well-organized π-conjugated self-assembled monolayer (SAM) at the interface. Such structural organization leads to the high optical emission efficiency of the RA SAM, even at room temperature. Additionally, photo-assisted electrical force microscopy, photo-assisted scanning Kelvin probe microscopy and Raman spectroscopy indicate a RA-induced graphene doping and photo-charge generation. Finally, the optical excitation of the RA monolayer generates surface potential changes on the hybrid system. In summary, interface-induced organized structures atop 2D materials may have an important impact on both design and operation of π-conjugated nanomaterial-based hybrid systems. PMID:29600157

Localization microscopy of DNA in situ using Vybrant(®) DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution.

PubMed

Żurek-Biesiada, Dominika; Szczurek, Aleksander T; Prakash, Kirti; Mohana, Giriram K; Lee, Hyun-Keun; Roignant, Jean-Yves; Birk, Udo J; Dobrucki, Jurek W; Cremer, Christoph

2016-05-01

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant(®) DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei of fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10(6) signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy. Copyright © 2016. Published by Elsevier Inc.

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

NASA Astrophysics Data System (ADS)

Skala, Melissa Caroline

2007-12-01

Cancer morbidity and mortality is greatly reduced when the disease is diagnosed and treated early in its development. Tissue biopsies are the gold standard for cancer diagnosis, and an accurate diagnosis requires a biopsy from the malignant portion of an organ. Light, guided through a fiber optic probe, could be used to inspect regions of interest and provide real-time feedback to determine the optimal tissue site for biopsy. This approach could increase the diagnostic accuracy of current biopsy procedures. The studies in this thesis have characterized changes in tissue optical signals with carcinogenesis, increasing our understanding of the sensitivity of optical techniques for cancer detection. All in vivo studies were conducted on the dimethylbenz[alpha]anthracene treated hamster cheek pouch model of epithelial carcinogenesis. Multiphoton microscopy studies in the near infrared wavelength region quantified changes in tissue morphology and fluorescence with carcinogenesis in vivo. Statistically significant morphological changes with precancer included increased epithelial thickness, loss of stratification in the epithelium, and increased nuclear diameter. Fluorescence changes included a statistically significant decrease in the epithelial fluorescence intensity per voxel at 780 nm excitation, a decrease in the fluorescence lifetime of protein-bound nicotinamide adenine dinucleotide (NADH, an electron donor in oxidative phosphorylation), and an increase in the fluorescence lifetime of protein-bound flavin adenine dinucleotide (FAD, an electron acceptor in oxidative phosphorylation) with precancer. The redox ratio (fluorescence intensity of FAD/NADH, a measure of the cellular oxidation-reduction state) did not significantly change with precancer. Cell culture experiments (MCF10A cells) indicated that the decrease in protein-bound NADH with precancer could be due to increased levels of glycolysis. Point measurements of diffuse reflectance and fluorescence spectra in the ultraviolet to visible wavelength range indicated that the most diagnostic optical signals originate from sub-surface tissue layers. Optical properties extracted from these spectroscopy measurements showed a significant decrease in the hemoglobin saturation, absorption coefficient, reduced scattering coefficient and fluorescence intensity (at 400 nm excitation) in neoplastic compared to normal tissues. The results from these studies indicate that multiphoton microscopy and optical spectroscopy can non-invasively provide information on tissue structure and function in vivo that is related to tissue pathology.

Time-resolved quantitative-phase microscopy of laser-material interactions using a wavefront sensor.

PubMed

Gallais, Laurent; Monneret, Serge

2016-07-15

We report on a simple and efficient technique based on a wavefront sensor to obtain time-resolved amplitude and phase images of laser-material interactions. The main interest of the technique is to obtain quantitative self-calibrated phase measurements in one shot at the femtosecond time-scale, with high spatial resolution. The technique is used for direct observation and quantitative measurement of the Kerr effect in a fused silica substrate and free electron generation by photo-ionization processes in an optical coating.

Structure and constitution of glass and steel compound in glass-metal composite

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

Lyubimova, Olga N.; Morkovin, Andrey V.; Dryuk, Sergey A.

2014-11-14

The research using methods of optical and scanning electronic microscopy was conducted and it discovered common factors on structures and diffusing zone forming after welding glass C49-1 and steel Ct3sp in technological process of creating new glass-metal composite. Different technological modes of steel surface preliminary oxidation welded with and without glass were investigated. The time of welding was varied from minimum encountering time to the time of stabilizing width of diffusion zone.

Holographic techniques for cellular fluorescence microscopy

NASA Astrophysics Data System (ADS)

Kim, Myung K.

2017-04-01

We have constructed a prototype instrument for holographic fluorescence microscopy (HFM) based on self-interference incoherent digital holography (SIDH) and demonstrate novel imaging capabilities such as differential 3D fluorescence microscopy and optical sectioning by compressive sensing.

Metal-Coated Optical Fibers for High Temperature Applications

NASA Technical Reports Server (NTRS)

Zeakes, Jason; Murphy, Kent; Claus, Richard; Greene, Jonathan; Tran, Tuan

1996-01-01

A DC magnetron sputtering system has been used to actively coat optical fibers with hermetic metal coatings during the fiber draw process. Thin films of Inconel 625 have been deposited on optical fibers and annealed in air at 2000 F. Scanning electron microscopy and Auger electron microscopy have been used to investigate the morphology and composition of the films prior to and following thermal cycling. Issues to be addressed include film adhesion, other coating materials, and a discussion of additional applications for this novel technology.

Rat brain imaging using full field optical coherence microscopy with short multimode fiber probe

NASA Astrophysics Data System (ADS)

Sato, Manabu; Saito, Daisuke; Kurotani, Reiko; Abe, Hiroyuki; Kawauchi, Satoko; Sato, Shunichi; Nishidate, Izumi

2017-02-01

We demonstrated FF OCM(full field optical coherence microscopy) using an ultrathin forward-imaging SMMF (short multimode fiber) probe of 50 μm core diameter, 125 μm diameter, and 7.4 mm length, which is a typical graded-index multimode fiber for optical communications. The axial resolution was measured to be 2.20 μm, which is close to the calculated axial resolution of 2.06 μm. The lateral resolution was evaluated to be 4.38 μm using a test pattern. Assuming that the FWHM of the contrast is the DOF (depth of focus), the DOF of the signal is obtained at 36 μm and that of the OCM is 66 μm. The contrast of the OCT images was 6.1 times higher than that of the signal images due to the coherence gate. After an euthanasia the rat brain was resected and cut at 2.6mm tail from Bregma. Contacting SMMF to the primary somatosensory cortex and the agranular insular cortex of ex vivo brain, OCM images of the brain were measured 100 times with 2μm step. 3D OCM images of the brain were measured, and internal structure information was obtained. The feasibility of an SMMF as an ultrathin forward-imaging probe in full-field OCM has been demonstrated.

Effect of thickness on surface morphology, optical and humidity sensing properties of RF magnetron sputtered CCTO thin films

NASA Astrophysics Data System (ADS)

Ahmadipour, Mohsen; Ain, Mohd Fadzil; Ahmad, Zainal Arifin

2016-11-01

In this study, calcium copper titanate (CCTO) thin films were deposited on ITO substrates successfully by radio frequency (RF) magnetron sputtering method in argon atmosphere. The CCTO thin films present a polycrystalline, uniform and porous structure. The surface morphology, optical and humidity sensing properties of the synthesized CCTO thin films have been studied by X-ray diffraction (XRD), atomic force microscopy (AFM), field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDX), UV-vis spectrophotometer and current-voltage (I-V) analysis. XRD and AFM confirmed that the intensity of peaks and pore size of CCTO thin films were enhanced by increasing the thin films. Tauc plot method was adopted to estimate the optical band gaps. The surface structure and energy band gaps of the deposited films were affected by film thickness. Energy band gap of the layers were 3.76 eV, 3.68 eV and 3.5 eV for 200 nm, 400 nm, and 600 nm CCTO thin films layer, respectively. The humidity sensing properties were measured by using direct current (DC) analysis method. The response times were 12 s, 22 s, and 35 s while the recovery times were 500 s, 600 s, and 650 s for 200 nm, 400 nm, and 600 nm CCTO thin films, respectively at humidity range of 30-90% relative humidity (RH).

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.

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

PubMed Central

Neuman, Keir C.; Nagy, Attila

2012-01-01

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

Chemical Silver Coating of Fiber Tips in Near-Field Scanning Optical Microscopy

NASA Technical Reports Server (NTRS)

Vikram, Chandra S.; Witherow, William K.

1998-01-01

We report what is believed to be the first experimental demonstration of silver coating by a wet chemical process on tapered fiber tips used in near-field scanning optical microscopy. The process is at room temperature and pressure and takes only a few minutes to complete. Many tips can be simultaneously coated.

Quantitative and qualitative changes in teaching histology by means of virtual microscopy in an introductory course in human anatomy.

PubMed

Husmann, Polly R; O'Loughlin, Valerie Dean; Braun, Mark W

2009-10-01

This study compares overall laboratory averages and individual test scores along with a student survey to determine the effects of using virtual microscopy in place of optical microscopes in a large undergraduate human anatomy course. T-tests revealed that the first two laboratory examinations (of four) and the overall laboratory averages were significantly increased compared with the previous year. We hypothesize that this is due to students' ability to use and understand the technology quickly as opposed to learning how to maneuver an optical microscope. Students also responded positively in a survey about the virtual microscope, indicating that increased accessibility, ease of use, and the ability to understand the material were important components of the virtual microscope. In addition, an increase in student collaboration was noted because multiple students were able to view the image at a time. This level of acceptance of virtual microscopy has been reported in previous studies, though this level of increased examination scores is rare. We attribute this to differences between the medical students, with whom this technology has been researched in the past, and undergraduate introductory students.

Motion-artifact-robust, polarization-resolved second-harmonic-generation microscopy based on rapid polarization switching with electro-optic Pockells cell and its application to in vivo visualization of collagen fiber orientation in human facial skin

PubMed Central

Tanaka, Yuji; Hase, Eiji; Fukushima, Shuichiro; Ogura, Yuki; Yamashita, Toyonobu; Hirao, Tetsuji; Araki, Tsutomu; Yasui, Takeshi

2014-01-01

Polarization-resolved second-harmonic-generation (PR-SHG) microscopy is a powerful tool for investigating collagen fiber orientation quantitatively with low invasiveness. However, the waiting time for the mechanical polarization rotation makes it too sensitive to motion artifacts and hence has hampered its use in various applications in vivo. In the work described in this article, we constructed a motion-artifact-robust, PR-SHG microscope based on rapid polarization switching at every pixel with an electro-optic Pockells cell (PC) in synchronization with step-wise raster scanning of the focus spot and alternate data acquisition of a vertical-polarization-resolved SHG signal and a horizontal-polarization-resolved one. The constructed PC-based PR-SHG microscope enabled us to visualize orientation mapping of dermal collagen fiber in human facial skin in vivo without the influence of motion artifacts. Furthermore, it implied the location and/or age dependence of the collagen fiber orientation in human facial skin. The robustness to motion artifacts in the collagen orientation measurement will expand the application scope of SHG microscopy in dermatology and collagen-related fields. PMID:24761292

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.

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.

Non-Linear Optical Microscopy Sheds Light on Cardiovascular Disease

PubMed Central

Caorsi, Valentina; Toepfer, Christopher; Sikkel, Markus B.; Lyon, Alexander R.; MacLeod, Ken; Ferenczi, Mike A.

2013-01-01

Many cardiac diseases have been associated with increased fibrosis and changes in the organization of fibrillar collagen. The degree of fibrosis is routinely analyzed with invasive histological and immunohistochemical methods, giving a limited and qualitative understanding of the tissue's morphological adaptation to disease. Our aim is to quantitatively evaluate the increase in fibrosis by three-dimensional imaging of the collagen network in the myocardium using the non-linear optical microscopy techniques Two-Photon Excitation microscopy (TPE) and Second Harmonic signal Generation (SHG). No sample staining is needed because numerous endogenous fluorophores are excited by a two-photon mechanism and highly non-centrosymmetric structures such as collagen generate strong second harmonic signals. We propose for the first time a 3D quantitative analysis to carefully evaluate the increased fibrosis in tissue from a rat model of heart failure post myocardial infarction. We show how to measure changes in fibrosis from the backward SHG (BSHG) alone, as only backward-propagating SHG is accessible for true in vivo applications. A 5-fold increase in collagen I fibrosis is detected in the remote surviving myocardium measured 20 weeks after infarction. The spatial distribution is also shown to change markedly, providing insight into the morphology of disease progression. PMID:23409139

Visualizing radiofrequency-skin interaction using multiphoton microscopy in vivo.

PubMed

Tsai, Tsung-Hua; Lin, Sung-Jan; Lee, Woan-Ruoh; Wang, Chun-Chin; Hsu, Chih-Ting; Chu, Thomas; Dong, Chen-Yuan

2012-02-01

Redundant skin laxity is a major feature of aging. Recently, radiofrequency has been introduced for nonablative tissue tightening by volumetric heating of the deep dermis. Despite the wide range of application based on this therapy, the effect of this technique on tissue and the subsequent tissue remodeling have not been investigated in detail. Our objective is to evaluate the potential of non-linear optics, including multiphoton autofluorescence and second harmonic generation (SHG) microscopy, as a non-invasive imaging modality for the real-time study of radiofrequency-tissue interaction. Electro-optical synergy device (ELOS) was used as the radiofrequency source in this study. The back skin of nude mouse was irradiated with radiofrequency at different passes. We evaluated the effect on skin immediately and 1 month after treatment with multiphoton microscopy. Corresponding histology was performed for comparison. We found that SHG is negatively correlated to radiofrequency passes, which means that collagen structural disruption happens immediately after thermal damage. After 1 month of collagen remodeling, SHG signals increased above baseline, indicating that collagen regeneration has occurred. Our findings may explain mechanism of nonablative skin tightening and were supported by histological examinations. Our work showed that monitoring the dermal heating status of RF and following up the detailed process of tissue reaction can be imaged and quantified with multiphoton microscopy non-invasively in vivo. Copyright © 2011. Published by Elsevier Ireland Ltd.

eduSPIM: Light Sheet Microscopy in the Museum

PubMed Central

Schmid, Benjamin; Weber, Michael; Huisken, Jan

2016-01-01

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

Brain plasticity and functionality explored by nonlinear optical microscopy

NASA Astrophysics Data System (ADS)

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

2010-02-01

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

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.

Plant cell wall characterization using scanning probe microscopy techniques

PubMed Central

Yarbrough, John M; Himmel, Michael E; Ding, Shi-You

2009-01-01

Lignocellulosic biomass is today considered a promising renewable resource for bioenergy production. A combined chemical and biological process is currently under consideration for the conversion of polysaccharides from plant cell wall materials, mainly cellulose and hemicelluloses, to simple sugars that can be fermented to biofuels. Native plant cellulose forms nanometer-scale microfibrils that are embedded in a polymeric network of hemicelluloses, pectins, and lignins; this explains, in part, the recalcitrance of biomass to deconstruction. The chemical and structural characteristics of these plant cell wall constituents remain largely unknown today. Scanning probe microscopy techniques, particularly atomic force microscopy and its application in characterizing plant cell wall structure, are reviewed here. We also further discuss future developments based on scanning probe microscopy techniques that combine linear and nonlinear optical techniques to characterize plant cell wall nanometer-scale structures, specifically apertureless near-field scanning optical microscopy and coherent anti-Stokes Raman scattering microscopy. PMID:19703302

High-speed laser microsurgery of alert fruit flies for fluorescence imaging of neural activity

PubMed Central

Sinha, Supriyo; Liang, Liang; Ho, Eric T. W.; Urbanek, Karel E.; Luo, Liqun; Baer, Thomas M.; Schnitzer, Mark J.

2013-01-01

Intravital microscopy is a key means of monitoring cellular function in live organisms, but surgical preparation of a live animal for microscopy often is time-consuming, requires considerable skill, and limits experimental throughput. Here we introduce a spatially precise (<1-µm edge precision), high-speed (<1 s), largely automated, and economical protocol for microsurgical preparation of live animals for optical imaging. Using a 193-nm pulsed excimer laser and the fruit fly as a model, we created observation windows (12- to 350-µm diameters) in the exoskeleton. Through these windows we used two-photon microscopy to image odor-evoked Ca2+ signaling in projection neuron dendrites of the antennal lobe and Kenyon cells of the mushroom body. The impact of a laser-cut window on fly health appears to be substantially less than that of conventional manual dissection, for our imaging durations of up to 18 h were ∼5–20 times longer than prior in vivo microscopy studies of hand-dissected flies. This improvement will facilitate studies of numerous questions in neuroscience, such as those regarding neuronal plasticity or learning and memory. As a control, we used phototaxis as an exemplary complex behavior in flies and found that laser microsurgery is sufficiently gentle to leave it intact. To demonstrate that our techniques are applicable to other species, we created microsurgical openings in nematodes, ants, and the mouse cranium. In conjunction with emerging robotic methods for handling and mounting flies or other small organisms, our rapid, precisely controllable, and highly repeatable microsurgical techniques should enable automated, high-throughput preparation of live animals for optical experimentation. PMID:24167298

Optical resonance imaging: An optical analog to MRI with sub-diffraction-limited capabilities.

PubMed

Allodi, Marco A; Dahlberg, Peter D; Mazuski, Richard J; Davis, Hunter C; Otto, John P; Engel, Gregory S

2016-12-21

We propose here optical resonance imaging (ORI), a direct optical analog to magnetic resonance imaging (MRI). The proposed pulse sequence for ORI maps space to time and recovers an image from a heterodyne-detected third-order nonlinear photon echo measurement. As opposed to traditional photon echo measurements, the third pulse in the ORI pulse sequence has significant pulse-front tilt that acts as a temporal gradient. This gradient couples space to time by stimulating the emission of a photon echo signal from different lateral spatial locations of a sample at different times, providing a widefield ultrafast microscopy. We circumvent the diffraction limit of the optics by mapping the lateral spatial coordinate of the sample with the emission time of the signal, which can be measured to high precision using interferometric heterodyne detection. This technique is thus an optical analog of MRI, where magnetic-field gradients are used to localize the spin-echo emission to a point below the diffraction limit of the radio-frequency wave used. We calculate the expected ORI signal using 15 fs pulses and 87° of pulse-front tilt, collected using f /2 optics and find a two-point resolution 275 nm using 800 nm light that satisfies the Rayleigh criterion. We also derive a general equation for resolution in optical resonance imaging that indicates that there is a possibility of superresolution imaging using this technique. The photon echo sequence also enables spectroscopic determination of the input and output energy. The technique thus correlates the input energy with the final position and energy of the exciton.

Dielectric Optical-Controllable Magnifying Lens by Nonlinear Negative Refraction

PubMed Central

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

2015-01-01

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

Applications of optically detected MRI for enhanced contrast and penetration in metal

NASA Astrophysics Data System (ADS)

Ruangchaithaweesuk, Songtham; Yu, Dindi S.; Garcia, Nissa C.; Yao, Li; Xu, Shoujun

2012-10-01

We report quantitative measurements using optically detected magnetic resonance imaging (MRI) for enhanced pH contrast and flow inside porous metals. Using a gadolinium chelate as the pH contrast agent, we show the response is 0.6 s-1 mM-1 per pH unit at the ambient magnetic field for the pH range 6-8.5. A stopped flow scheme was used to directly measure T1 relaxation time to determine the relaxivity. Flow profiles and images were obtained for a series of porous metals with different average pore sizes. The signal amplitudes and spatial distributions were compared. A clogged region in one of the samples was revealed using optically detected MRI but not optical imaging or scanning electron microscopy. These applications will significantly broaden the impact of optically detected MRI in chemical imaging and materials research.

Fabrication of a Porous Fiber Cladding Material Using Microsphere Templating for Improved Response Time with Fiber Optic Sensor Arrays

PubMed Central

Henning, Paul E.; Rigo, M. Veronica; Geissinger, Peter

2012-01-01

A highly porous optical-fiber cladding was developed for evanescent-wave fiber sensors, which contains sensor molecules, maintains guiding conditions in the optical fiber, and is suitable for sensing in aqueous environments. To make the cladding material (a poly(ethylene) glycol diacrylate (PEGDA) polymer) highly porous, a microsphere templating strategy was employed. The resulting pore network increases transport of the target analyte to the sensor molecules located in the cladding, which improves the sensor response time. This was demonstrated using fluorescein-based pH sensor molecules, which were covalently attached to the cladding material. Scanning electron microscopy was used to examine the structure of the templated polymer and the large network of interconnected pores. Fluorescence measurements showed a tenfold improvement in the response time for the templated polymer and a reliable pH response over a pH range of five to nine with an estimated accuracy of 0.08 pH units. PMID:22654644

Photo-induced changes in nano-copper oxide for optoelectronic applications

NASA Astrophysics Data System (ADS)

Hendi, A. A.; Rashad, M.

2018-06-01

Copper oxide (CuO) nanoparticles (NPs) have been prepared using microwave irradiation. A mother material was copper nitrate in distilled water. X-ray diffraction (XRD) and transmission electron microscopy (TEM) were used for characterizing the NPs powders. Thermal Gravimetric Analysis (TGA) and Differential Thermal Analysis (DTA) were measured for as-prepared CuO NPs. The obtained oxides NPs were confirmed produced during chemical precipitation by these characterizions. These NPs were dropped on top of glass substrate for measuring the optical characterizions. Both linear and nonlinear optical properties of the as-prepared CuO NP films were studied. The optical energy gap of the as-prepared CuO NP films is equal to 3.98 eV, which is higher than that of the bulk material. The effect of ultraviolet (UV) light irradiation on the CuO NP films was investigated at 2 and 5 h for study the photo-induced effect. The optical properties of CuO NP films were measured as a function of these UV irradiation time. The optical constants for as-prepared and irradiated CuO NP films were calculated which reflect the affect of UV irradiation time. As observed from these optical results, a highly forced for optoelectronic applications.

Optical sensor based on a single CdS nanobelt.

PubMed

Li, Lei; Yang, Shuming; Han, Feng; Wang, Liangjun; Zhang, Xiaotong; Jiang, Zhuangde; Pan, Anlian

2014-04-23

In this paper, an optical sensor based on a cadmium sulfide (CdS) nanobelt has been developed. The CdS nanobelt was synthesized by the vapor phase transportation (VPT) method. X-Ray Diffraction (XRD) and Transmission Electron Microscopy (TEM) results revealed that the nanobelt had a hexagonal wurtzite structure of CdS and presented good crystal quality. A single nanobelt Schottky contact optical sensor was fabricated by the electron beam lithography (EBL) technique, and the device current-voltage results showed back-to-back Schottky diode characteristics. The photosensitivity, dark current and the decay time of the sensor were 4 × 10⁴, 31 ms and 0.2 pA, respectively. The high photosensitivity and the short decay time were because of the exponential dependence of photocurrent on the number of the surface charges and the configuration of the back to back Schottky junctions.

Rich stochastic dynamics of co-doped Er:Yb fluorescence upconversion nanoparticles in the presence of thermal, non-conservative, harmonic and optical forces

NASA Astrophysics Data System (ADS)

Nome, Rene A.; Sorbello, Cecilia; Jobbágy, Matías; Barja, Beatriz C.; Sanches, Vitor; Cruz, Joyce S.; Aguiar, Vinicius F.

2017-03-01

The stochastic dynamics of individual co-doped Er:Yb upconversion nanoparticles (UCNP) were investigated from experiments and simulations. The UCNP were characterized by high-resolution scanning electron microscopy, dynamic light scattering, and zeta potential measurements. Single UCNP measurements were performed by fluorescence upconversion micro-spectroscopy and optical trapping. The mean-square displacement (MSD) from single UCNP exhibited a time-dependent diffusion coefficient which was compared with Brownian dynamics simulations of a viscoelastic model of harmonically bound spheres. Experimental time-dependent two-dimensional trajectories of individual UCNP revealed correlated two-dimensional nanoparticle motion. The measurements were compared with stochastic trajectories calculated in the presence of a non-conservative rotational force field. Overall, the complex interplay of UCNP adhesion, thermal fluctuations and optical forces led to a rich stochastic behavior of these nanoparticles.

Ultrafast Imaging of Chiral Surface Plasmon by Photoemission Electron Microscopy

NASA Astrophysics Data System (ADS)

Dai, Yanan; Dabrowski, Maciej; Petek, Hrvoje

We employ Time-Resolved Photoemission Electron Microscopy (TR-PEEM) to study surface plasmon polariton (SPP) wave packet dynamics launched by tunable (VIS-UV) femtosecond pulses of various linear and circular polarizations. The plasmonic structures are micron size single-crystalline Ag islands grown in situ on Si surfaces and characterized by Low Energy Electron Microscopy (LEEM). The local fields of plasmonic modes enhance two and three photon photoemission (2PP and 3PP) at the regions of strong field enhancement. Imaging of the photoemission signal with PEEM electron optics thus images the plasmonic fields excited in the samples. The observed PEEM images with left and right circularly polarized light show chiral images, which is a consequence of the transverse spin momentum of surface plasmon. By changing incident light polarization, the plasmon interference pattern shifts with light ellipticity indicating a polarization dependent excitation phase of SPP. In addition, interferometric-time resolved measurements record the asymmetric SPP wave packet motion in order to characterize the dynamical properties of chiral SPP wave packets.

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.

Handheld optical-resolution photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Lin, Li; Zhang, Pengfei; Xu, Song; Shi, Junhui; Li, Lei; Yao, Junjie; Wang, Lidai; Zou, Jun; Wang, Lihong V.

2017-04-01

Optical-resolution photoacoustic microscopy (OR-PAM) offers label-free in vivo imaging with high spatial resolution by acoustically detecting optical absorption contrasts via the photoacoustic effect. We developed a compact handheld OR-PAM probe for fast photoacoustic imaging. Different from benchtop microscopes, the handheld probe provides flexibility in imaging various anatomical sites. Resembling a cup in size, the probe uses a two-axis water-immersible microelectromechanical system mirror to scan both the illuminating optical beam and resultant acoustic beam. The system performance was tested in vivo by imaging the capillary bed in a mouse ear and both the capillary bed and a mole on a human volunteer.

Evanescent Waves in High Numerical Aperture Aplanatic Solid Immersion Microscopy: Effects of Forbidden Light on Subsurface Imaging (Open Access, Publisher’s Version)

DTIC Science & Technology

2014-03-24

of the aSIL microscopy for semiconductor failure analysis and is applicable to imaging in quantum optics [18], biophotonics [19] and metrology [20...is usually of interest, the model can be adapted to applications in fields such as quantum optics and biophotonics for which the non-resonant

Emerging optical nanoscopy techniques

PubMed Central

Montgomery, Paul C; Leong-Hoi, Audrey

2015-01-01

To face the challenges of modern health care, new imaging techniques with subcellular resolution or detection over wide fields are required. Far field optical nanoscopy presents many new solutions, providing high resolution or detection at high speed. We present a new classification scheme to help appreciate the growing number of optical nanoscopy techniques. We underline an important distinction between superresolution techniques that provide improved resolving power and nanodetection techniques for characterizing unresolved nanostructures. Some of the emerging techniques within these two categories are highlighted with applications in biophysics and medicine. Recent techniques employing wider angle imaging by digital holography and scattering lens microscopy allow superresolution to be achieved for subcellular and even in vivo, imaging without labeling. Nanodetection techniques are divided into four subcategories using contrast, phase, deconvolution, and nanomarkers. Contrast enhancement is illustrated by means of a polarized light-based technique and with strobed phase-contrast microscopy to reveal nanostructures. Very high sensitivity phase measurement using interference microscopy is shown to provide nanometric surface roughness measurement or to reveal internal nanometric structures. Finally, the use of nanomarkers is illustrated with stochastic fluorescence microscopy for mapping intracellular structures. We also present some of the future perspectives of optical nanoscopy. PMID:26491270

Multiple speckle illumination for optical-resolution photoacoustic imaging

NASA Astrophysics Data System (ADS)

Poisson, Florian; Stasio, Nicolino; Moser, Christophe; Psaltis, Demetri; Bossy, Emmanuel

2017-03-01

Optical-resolution photoacoustic microscopy offers exquisite and specific contrast to optical absorption. Conventional approaches generally involves raster scanning a focused spot over the sample. Here, we demonstrate that a full-field illumination approach with multiple speckle illumination can also provide diffraction-limited optical-resolution photoacoustic images. Two different proof-of-concepts are demonstrated with micro-structured test samples. The first approach follows the principle of correlation/ghost imaging,1, 2 and is based on cross-correlating photoacoustic signals under multiple speckle illumination with known speckle patterns measured during a calibration step. The second approach is a speckle scanning microscopy technique, which adapts the technique proposed in fluorescence microscopy by Bertolotti and al.:3 in our work, spatially unresolved photoacoustic measurements are performed for various translations of unknown speckle patterns. A phase-retrieval algorithm is used to reconstruct the object from the knowledge of the modulus of its Fourier Transform yielded by the measurements. Because speckle patterns naturally appear in many various situations, including propagation through biological tissue or multi-mode fibers (for which focusing light is either very demanding if not impossible), speckle-illumination-based photoacoustic microscopy provides a powerful framework for the development of novel reconstruction approaches, well-suited to compressed sensing approaches.2

Tip/tilt-compensated through-focus scanning optical microscopy

NASA Astrophysics Data System (ADS)

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

2016-11-01

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

Optical imaging modalities: From design to diagnosis of skin cancer

NASA Astrophysics Data System (ADS)

Korde, Vrushali Raj

This study investigates three high resolution optical imaging modalities to better detect and diagnose skin cancer. The ideal high resolution optical imaging system can visualize pre-malignant tissue growth non-invasively with resolution comparable to histology. I examined 3 modalities which approached this goal. The first method examined was high magnification microscopy of thin stained tissue sections, together with a statistical analysis of nuclear chromatin patterns termed Karyometry. This method has subcellular resolution, but it necessitates taking a biopsy at the desired tissue site and imaging the tissue ex-vivo. My part of this study was to develop an automated nuclear segmentation algorithm to segment cell nuclei in skin histology images for karyometric analysis. The results of this algorithm were compared to hand segmented cell nuclei in the same images, and it was concluded that the automated segmentations can be used for karyometric analysis. The second optical imaging modality I investigated was Optical Coherence Tomography (OCT). OCT is analogous to ultrasound, in which sound waves are delivered into the body and the echo time and reflected signal magnitude are measured. Due to the fast speed of light and detector temporal integration times, low coherence interferometry is needed to gate the backscattered light. OCT acquires cross sectional images, and has an axial resolution of 1-15 mum (depending on the source bandwidth) and a lateral resolution of 10-20 mum (depending on the sample arm optics). While it is not capable of achieving subcellular resolution, it is a non-invasive imaging modality. OCT was used in this study to evaluate skin along a continuum from normal to sun damaged to precancer. I developed algorithms to detect statistically significant differences between images of sun protected and sun damaged skin, as well as between undiseased and precancerous skin. An Optical Coherence Microscopy (OCM) endoscope was developed in the third portion of this study. OCM is a high resolution en-face imaging modality. It is a hybrid system that combines the principles of confocal microscopy with coherence gating to provide an increased imaging depth. It can also be described as an OCT system with a high NA objective. Similar to OCT, the axial resolution is determined by the source center wavelength and bandwidth. The NA of the sample arm optics determines the lateral resolution, usually on the order of 1-5 mum. My effort on this system was to develop a handheld endoscope. To my knowledge, an OCM endoscope has not been developed prior to this work. An image of skin was taken as a proof of concept. This rigid handheld OCM endoscope will be useful for applications ranging from minimally invasive surgical imaging to non-invasively assessing dysplasia and sun damage in skin.

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

PubMed

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

2013-11-01

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

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

PubMed Central

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

2012-01-01

In vivo optical microscopic imaging techniques have recently emerged as important tools for the study of neurobiological development and pathophysiology. In particular, two-photon microscopy has proved to be a robust and highly flexible method for in vivo imaging in highly scattering tissue. However, two-photon imaging typically requires extrinsic dyes or contrast agents, and imaging depths are limited to a few hundred microns. Here we demonstrate Optical Coherence Microscopy (OCM) for in vivo imaging of neuronal cell bodies and cortical myelination up to depths of ~1.3 mm in the rat neocortex. Imaging does not require the administration of exogenous dyes or contrast agents, and is achieved through intrinsic scattering contrast and image processing alone. Furthermore, using OCM we demonstrate in vivo, quantitative measurements of optical properties (index of refraction and attenuation coefficient) in the cortex, and correlate these properties with laminar cellular architecture determined from the images. Lastly, we show that OCM enables direct visualization of cellular changes during cell depolarization and may therefore provide novel optical markers of cell viability. PMID:22330462

Stroboscobic near-field scanning optical microscopy for 3D mapping of mode profiles of plasmonic nanostructures (Conference Presentation)

NASA Astrophysics Data System (ADS)

Dana, Aykutlu; Ozgur, Erol; Torunoglu, Gamze

2016-09-01

We present a dynamic approach to scanning near field optical microscopy that extends the measurement technique to the third dimension, by strobing the illumination in sync with the cantilever oscillation. Nitrogen vacancy (NV) centers in nanodiamonds placed on cantilever tips are used as stable emitters for emission enhancement. Local field enhancement and modulation of optical density states are mapped in three dimensions based on fluorescence intensity and spectrum changes as the tip is scanned over plasmonic nanostructures. The excitation of NV centers is done using a total internal reflection setup. Using a digital phase locked loop to pulse the excitation in various tip sample separations, 2D slices of fluorescence enhancement can be recorded. Alternatively, a conventional SNOM tip can be used to selectively couple wideband excitation to the collection path, with subdiffraction resolution of 60 nm in x and y and 10 nm in z directions. The approach solves the problem of tip-sample separation stabilization over extended periods of measurement time, required to collect data resolved in emission wavelength and three spatial dimensions. The method can provide a unique way of accessing the three dimensional field and mode profiles of nanophotonics structures.

Biomechanics and dynamics of red blood cells probed by optical tweezers and digital holographic microscopy

NASA Astrophysics Data System (ADS)

Cardenas, Nelson; Thomas, Pattrick; Yu, Lingfeng; Mohanty, Samarendra

2011-03-01

Red blood cells (RBC), with their unique viscoelastic properties, can undergo large deformations during interaction with fluid flow and migration through narrow capillaries. Both local and overall viscoelastic property is important for cellular function and change in these properties indicate diseased condition. Though biomechanics of the cells have been studied using variety of physical techniques (AFM, optically-trapped anchoring beads and microcapilary aspiration) in force regime 10pN, little is studied at low force regime <1pN. Such perturbations are not only hard to exercise on the cell membrane, but quantification of such deformations becomes extremely difficult. By application of low power optical tweezers directly on cell membrane, we could locally perturb discotic RBC along the axial direction, which was monitored dynamically by digital holographic microscopy-a real time, wide-field imaging method having nm axial resolution. The viscoelastic property of the RBC at low force regime was found to be significantly different from that of high-force regime. The results were found to be in good agreement with the simulation results obtained using finite element model of the axially-stretched RBC. The simulations and results of viscoelestic measurements will be presented.

Applicability of confocal laser scanning microscopy for evaluation and monitoring of cutaneous wound healing

NASA Astrophysics Data System (ADS)

Lange-Asschenfeldt, Susanne; Bob, Adrienne; Terhorst, Dorothea; Ulrich, Martina; Fluhr, Joachim; Mendez, Gil; Roewert-Huber, Hans-Joachim; Stockfleth, Eggert; Lange-Asschenfeldt, Bernhard

2012-07-01

There is a high demand for noninvasive imaging techniques for wound assessment. In vivo reflectance confocal laser scanning microscopy (CLSM) represents an innovative optical technique for noninvasive evaluation of normal and diseased skin in vivo at near cellular resolution. This study was designed to test the feasibility of CLSM for noninvasive analysis of cutaneous wound healing in 15 patients (7 male/8 female), including acute and chronic, superficial and deep dermal skin wounds. A commercially available CLSM system was used for the assessment of wound bed and wound margins in order to obtain descriptive cellular and morphological parameters of cutaneous wound repair noninvasively and over time. CLSM was able to visualize features of cutaneous wound repair in epidermal and superficial dermal wounds, including aspects of inflammation, neovascularisation, and tissue remodelling in vivo. Limitations include the lack of mechanic fixation of the optical system on moist surfaces restricting the analysis of chronic skin wounds to the wound margins, as well as a limited optical resolution in areas of significant slough formation. By describing CLSM features of cutaneous inflammation, vascularisation, and epithelialisation, the findings of this study support the role of CLSM in modern wound research and management.

Investigating Molecular Level Stress-Strain Relationships in Entangled F-Actin Networks by Combined Force-Measuring Optical Tweezers and Fluorescence Microscopy

NASA Astrophysics Data System (ADS)

Lee, Kent; Henze, Dean; Robertson-Anderson, Rae

2013-03-01

Actin is an important cytoskeletal protein involved in cell structure and motility, cancer invasion and metastasis, and muscle contraction. The intricate viscoelastic properties of filamentous actin (F-actin) networks allow for the many dynamic roles of actin, thus warranting investigation. Exploration of this unique stress-strain/strain-rate relationship in complex F-actin networks can also improve biomimetic materials engineering. Here, we use optical tweezers with fluorescence microscopy to study the viscoelastic properties of F-actin networks on the microscopic level. Optically trapped microspheres embedded in various F-actin networks are moved through the network using a nanoprecision piezoelectric stage. The force exerted on the microspheres by the F-actin network and subsequent force relaxation are measured, while a fraction of the filaments in the network are fluorescent-labeled to observe filament deformation in real-time. The dependence of the viscoelastic properties of the network on strain rates and amplitudes as well as F-actin concentration is quantified. This approach provides the much-needed link between induced force and deformation over localized regimes (tens of microns) and down to the single molecule level.

Synthesis and characterization of metal-dielectric composites with copper nanoparticles embedded in a glass matrix: A multitechnique approach

NASA Astrophysics Data System (ADS)

Lipinska-Kalita, Kristina E.; Krol, Denise M.; Hemley, Russell J.; Mariotto, Gino; Kalita, Patricia E.; Ohki, Yoshimichi

2005-09-01

The precipitation and growth of copper nanoparticles in an optically transparent aluminosilicate glass matrix was investigated. The size of particles in this heterophase glass-based composite was modified in a controlled manner by isothermal heat treatments. A multitechnique approach, consisting of Raman scattering spectroscopy, high-resolution transmission electron microscopy, x-ray diffraction technique, and optical absorption spectroscopy, has been used to study the nucleation and crystallization processes. Optical absorption spectroscopy revealed the presence of intense absorption bands attributed to oscillations of free electrons, known as the surface-plasmon resonance band of copper particles, and confirmed a gradual increase of the particles' mean size and density with annealing time. The Raman scattering on acoustical phonons from Cu quantum dots in the glass matrix measured for off-resonance conditions demonstrated the presence of intense, inhomogeneously broadened peaks that have been assigned to the confined acoustic eigenmodes of copper nanoparticles. The particle-size dependence of the acoustic peak energies and the relation between the size distribution and bandwidths of these peaks were derived. High-resolution transmission electron microscopy was used to monitor the nucleation of the nanoparticles and to estimate their mean size.

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

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.

Towards non-invasive 3D hepatotoxicity assays with optical coherence phase microscopy

NASA Astrophysics Data System (ADS)

Nelson, Leonard J.; Koulovasilopoulos, Andreas; Treskes, Philipp; Hayes, Peter C.; Plevris, John N.; Bagnaninchi, Pierre O.

2015-03-01

Three-dimensional tissue-engineered models are increasingly recognised as more physiologically-relevant than standard 2D cell culture for pre-clinical drug toxicity testing. However, many types of conventional toxicity assays are incompatible with dense 3D tissues. This study investigated the use of optical coherence phase microscopy (OCPM) as a novel approach to assess cell death in 3D tissue culture. For 3D micro-spheroid formation Human hepatic C3A cells were encapsulated in hyaluronic acid gels and cultured in 100μl MEME/10%FBS in 96-well plates. After spheroid formation the 3D liver constructs were exposed to acetaminophen on culture day 8. Acetaminophen hepatotoxicity in 3D cultures was evaluated using standard biochemical assays. An inverted OCPM in common path configuration was developed with a Callisto OCT engine (Thorlabs), centred at 930nm and a custom scanning head. Intensity data were used to perform in-depth microstructural imaging. In addition, phase fluctuations were measured by collecting several successive B scans at the same location, and statistics on the first time derivative of the phase, i.e. time fluctuations, were analysed over the acquisition time interval to retrieve overall cell viability. OCPM intensity (cell cluster size) and phase fluctuation statistics were directly compared with biochemical assays. In this study, we investigated optical coherence phase tomography to assess cell death in a 3d liver model after exposure to a prototypical hepatotoxin, acetaminophen. We showed that OCPM has the potential to assess noninvasively and label-free drug toxicity in 3D tissue models.

Damage mechanisms of MoN/SiN multilayer optics for next-generation pulsed XUV light sources.

PubMed

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

2011-01-03

We investigated the damage mechanism of MoN/SiN multilayer XUV optics under two extreme conditions: thermal annealing and irradiation with single shot intense XUV pulses from the free-electron laser facility in Hamburg - FLASH. The damage was studied "post-mortem" by means of X-ray diffraction, interference-polarizing optical microscopy, atomic force microscopy, and scanning transmission electron microscopy. Although the timescale of the damage processes and the damage threshold temperatures were different (in the case of annealing it was the dissociation temperature of Mo2N and in the case of XUV irradiation it was the melting temperature of MoN) the main damage mechanism is very similar: molecular dissociation and the formation of N2, leading to bubbles inside the multilayer structure.

Multimodal nonlinear imaging of arabidopsis thaliana root cell

NASA Astrophysics Data System (ADS)

Jang, Bumjoon; Lee, Sung-Ho; Woo, Sooah; Park, Jong-Hyun; Lee, Myeong Min; Park, Seung-Han

2017-07-01

Nonlinear optical microscopy has enabled the possibility to explore inside the living organisms. It utilizes ultrashort laser pulse with long wavelength (greater than 800nm). Ultrashort pulse produces high peak power to induce nonlinear optical phenomenon such as two-photon excitation fluorescence (TPEF) and harmonic generations in the medium while maintaining relatively low average energy pre area. In plant developmental biology, confocal microscopy is widely used in plant cell imaging after the development of biological fluorescence labels in mid-1990s. However, fluorescence labeling itself affects the sample and the sample deviates from intact condition especially when labelling the entire cell. In this work, we report the dynamic images of Arabidopsis thaliana root cells. This demonstrates the multimodal nonlinear optical microscopy is an effective tool for long-term plant cell imaging.

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

NASA Astrophysics Data System (ADS)

DeArmond, Fredrick Michael

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

Fabrication and characterization of optical-fiber nanoprobes for scanning near-field optical microscopy.

PubMed

Essaidi, N; Chen, Y; Kottler, V; Cambril, E; Mayeux, C; Ronarch, N; Vieu, C

1998-02-01

The current scanning near-field optical microscopy has been developed with optical-fiber probes obtained by use of either laser-heated pulling or chemical etching. For high-resolution near-field imaging, the detected signal is rapidly attenuated as the aperture size of the probe decreases. It is thus important to fabricate probes optimized for both spot size and optical transmission. We present a two-step fabrication that allowed us to achieve an improved performance of the optical-fiber probes. Initially, a CO(2) laser-heated pulling was used to produce a parabolic transitional taper ending with a top thin filament. Then, a rapid chemical etching with 50% buffered hydrofluoric acid was used to remove the thin filament and to result in a final conical tip on the top of the parabolic transitional taper. Systematically, we obtained optical-fiber nanoprobes with the apex size as small as 10 nm and the final cone angle varying from 15 degrees to 80 degrees . It was found that the optical transmission efficiency increases rapidly as the taper angle increases from 15 degrees to 50 degrees , but a further increase in the taper angle gives rise to important broadening of the spot size. Finally, the fabricated nanoprobes were used in photon-scanning tunneling microscopy, which allowed observation of etched double lines and grating structures with periods as small as 200 nm.

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

PubMed

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

2006-07-01

In recent years, the study of single biomolecules using fluorescence microscopy and atomic force microscopy (AFM) techniques has resulted in a plethora of new information regarding the physics underlying these complex biological systems. It is especially advantageous to be able to measure the optical, topographical, and mechanical properties of single molecules simultaneously. Here an AFM is used that is especially designed for integration with an inverted optical microscope and that has a near-infrared light source (850 nm) to eliminate interference between the optical experiment and the AFM operation. The Tip Assisted Optics (TAO) system consists of an additional 100 x 100-microm(2) X-Y scanner for the sample, which can be independently and simultaneously used with the AFM scanner. This allows the offset to be removed between the confocal optical image obtained with the sample scanner and the simultaneously acquired AFM topography image. The tip can be positioned exactly into the optical focus while the user can still navigate within the AFM image for imaging or manipulation of the sample. Thus the tip-enhancement effect can be maximized and it becomes possible to perform single molecule manipulation experiments within the focus of a confocal optical image. Here this is applied to simultaneous measurement of single quantum dot fluorescence and topography with high spatial resolution. (c) 2006 Wiley Periodicals, Inc.

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

PubMed Central

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

2013-01-01

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

Surface Characterization.

ERIC Educational Resources Information Center

Fulghum, J. E.; And Others

1989-01-01

This review is divided into the following analytical methods: ion spectroscopy, electron spectroscopy, scanning tunneling microscopy, atomic force microscopy, optical spectroscopy, desorption techniques, and X-ray techniques. (MVL)

Optical tracking of embryonic vertebrates behavioural responses using automated time-resolved video-microscopy system

NASA Astrophysics Data System (ADS)

Walpitagama, Milanga; Kaslin, Jan; Nugegoda, Dayanthi; Wlodkowic, Donald

2016-12-01

The fish embryo toxicity (FET) biotest performed on embryos of zebrafish (Danio rerio) has gained significant popularity as a rapid and inexpensive alternative approach in chemical hazard and risk assessment. The FET was designed to evaluate acute toxicity on embryonic stages of fish exposed to the test chemical. The current standard, similar to most traditional methods for evaluating aquatic toxicity provides, however, little understanding of effects of environmentally relevant concentrations of chemical stressors. We postulate that significant environmental effects such as altered motor functions, physiological alterations reflected in heart rate, effects on development and reproduction can occur at sub-lethal concentrations well below than LC10. Behavioral studies can, therefore, provide a valuable integrative link between physiological and ecological effects. Despite the advantages of behavioral analysis development of behavioral toxicity, biotests is greatly hampered by the lack of dedicated laboratory automation, in particular, user-friendly and automated video microscopy systems. In this work we present a proof-of-concept development of an optical system capable of tracking embryonic vertebrates behavioral responses using automated and vastly miniaturized time-resolved video-microscopy. We have employed miniaturized CMOS cameras to perform high definition video recording and analysis of earliest vertebrate behavioral responses. The main objective was to develop a biocompatible embryo positioning structures that were suitable for high-throughput imaging as well as video capture and video analysis algorithms. This system should support the development of sub-lethal and behavioral markers for accelerated environmental monitoring.

Towards the use of bioresorbable fibers in time-domain diffuse optics.

PubMed

Di Sieno, Laura; Boetti, Nadia G; Dalla Mora, Alberto; Pugliese, Diego; Farina, Andrea; Konugolu Venkata Sekar, Sanathana; Ceci-Ginistrelli, Edoardo; Janner, Davide; Pifferi, Antonio; Milanese, Daniel

2018-01-01

In the last years bioresorbable materials are gaining increasing interest for building implantable optical components for medical devices. In this work we show the fabrication of bioresorbable optical fibers designed for diffuse optics applications, featuring large core diameter (up to 200 μm) and numerical aperture (0.17) to maximize the collection efficiency of diffused light. We demonstrate the suitability of bioresorbable fibers for time-domain diffuse optical spectroscopy firstly checking the intrinsic performances of the setup by acquiring the instrument response function. We then validate on phantoms the use of bioresorbable fibers by applying the MEDPHOT protocol to assess the performance of the system in measuring optical properties (namely, absorption and scattering coefficients) of homogeneous media. Further, we show an ex-vivo validation on a chicken breast by measuring the absorption and scattering spectra in the 500-1100 nm range using interstitially inserted bioresorbable fibers. This work represents a step toward a new way to look inside the body using optical fibers that can be implanted in patients. These fibers could be useful either for diagnostic (e. g. for monitoring the evolution after surgical interventions) or treatment (e. g. photodynamic therapy) purposes. Picture: Microscopy image of the 100 μm core bioresorbable fiber. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Solvothermally Synthesized Sb2Te3 Platelets Show Unexpected Optical Contrasts in Mid-Infrared Near-Field Scanning Microscopy.

PubMed

Hauer, Benedikt; Saltzmann, Tobias; Simon, Ulrich; Taubner, Thomas

2015-05-13

We report nanoscale-resolved optical investigations on the local material properties of Sb2Te3 hexagonal platelets grown by solvothermal synthesis. Using mid-infrared near-field microscopy, we find a highly symmetric pattern, which is correlated to a growth spiral and which extends over the entire platelet. As the origin of the optical contrast, we identify domains with different densities of charge carriers. On Sb2Te3 samples grown by other means, we did not find a comparable domain structure.

Giant optical field enhancement in multi-dielectric stacks by photon scanning tunneling microscopy

NASA Astrophysics Data System (ADS)

Ndiaye, C.; Zerrad, M.; Lereu, A. L.; Roche, R.; Dumas, Ph.; Lemarchand, F.; Amra, C.

2013-09-01

Dielectric optical thin films, as opposed to metallic, have been very sparsely explored as good candidates for absorption-based optical field enhancement. In such materials, the low imaginary part of the refractive index implies that absorption processes are usually not predominant. This leads to dielectric-based optical resonances mainly via waveguiding modes. We show here that when properly designed, a multi-layered dielectric thin films stack can give rise to optical resonances linked to total absorption. We report here, on such dielectric stack designed to possess a theoretical optical field enhancement above 1000. Using photon scanning tunneling microscopy, we experimentally evaluate the resulting field enhancement of the stack as well as the associated penetration depth. We thus demonstrate the capability of multi-dielectric stacks in generating giant optical field with tunable penetration depth (down to few dozens of nm).

An automated wide-field time-gated optically sectioning fluorescence lifetime imaging multiwell plate reader for high-content analysis of protein-protein interactions

NASA Astrophysics Data System (ADS)

Alibhai, Dominic; Kumar, Sunil; Kelly, Douglas; Warren, Sean; Alexandrov, Yuriy; Munro, Ian; McGinty, James; Talbot, Clifford; Murray, Edward J.; Stuhmeier, Frank; Neil, Mark A. A.; Dunsby, Chris; French, Paul M. W.

2011-03-01

We describe an optically-sectioned FLIM multiwell plate reader that combines Nipkow microscopy with wide-field time-gated FLIM, and its application to high content analysis of FRET. The system acquires sectioned FLIM images in <10 s/well, requiring only ~11 minutes to read a 96 well plate of live cells expressing fluorescent protein. It has been applied to study the formation of immature HIV virus like particles (VLPs) in live cells by monitoring Gag-Gag protein interactions using FLIM FRET of HIV-1 Gag transfected with CFP or YFP. VLP formation results in FRET between closely packed Gag proteins, as confirmed by our FLIM analysis that includes automatic image segmentation.

Apertureless near-field optical microscopy

NASA Astrophysics Data System (ADS)

Kazantsev, D. V.; Kuznetsov, E. V.; Timofeev, S. V.; Shelaev, A. V.; Kazantseva, E. A.

2017-05-01

We discuss the operating principles of the apertureless scanning near-field optical microscope (ASNOM), in which the probe acts as a rod antenna and its electromagnetic radiation plays the role of the registered signal. The phase and amplitude of the emitted wave vary depending on the ‘grounding conditions’ of the antenna tip at the sample point under study. Weak radiation from a tiny (2-15 μm long) tip is detected using optical homo- and heterodyning and the nonlinear dependence of the tip polarizability on the tip-surface distance. The lateral resolution of ASNOMs is determined by the tip curvature radius (1- 20 nm), regardless of the wavelength (500 nm-100 μm). ASNOMs are shown to be capable of providing a surface optical map with nanometer resolution and carrying out spectral- and time-resolved measurements at a selected point on the surface.

Composition, nanostructure, and optical properties of silver and silver-copper lusters

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

Pradell, Trinitat; Pavlov, Radostin S.; Carolina Gutierrez, Patricia

2012-09-01

Lusters are composite thin layers of coinage metal nanoparticles in glass displaying peculiar optical properties and obtained by a process involving ionic exchange, diffusion, and crystallization. In particular, the origin of the high reflectance (golden-shine) shown by those layers has been subject of some discussion. It has been attributed to either the presence of larger particles, thinner multiple layers or higher volume fraction of nanoparticles. The object of this paper is to clarify this for which a set of laboratory designed lusters are analysed by Rutherford backscattering spectroscopy, transmission electron microscopy, x-ray diffraction, and ultraviolet-visible spectroscopy. Model calculations and numericalmore » simulations using the finite difference time domain method were also performed to evaluate the optical properties. Finally, the correlation between synthesis conditions, nanostructure, and optical properties is obtained for these materials.« less

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

PubMed

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

2015-11-02

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

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

PubMed

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

2016-05-13

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

Special issue on high-resolution optical imaging

NASA Astrophysics Data System (ADS)

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

2013-09-01

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

Atomic force microscopy study of erythrocyte shape and membrane structure after treatment with a dihydropyridinic drug

NASA Astrophysics Data System (ADS)

Girasole, M.; Cricenti, A.; Generosi, R.; Congiu-Castellano, A.; Boffi, F.; Arcovito, A.; Boumis, G.; Amiconi, G.

2000-06-01

The overall shape and membrane surface of human erythrocytes (RBCs) in the presence of nifedipine (a dihydropyridinic drug used in the clinical treatment of hypertension and angina pectoris) were imaged by contact-mode atomic force microscopy. Nifedipine induces in RBCs relevant morphological changes the extent of which increases as a function of drug concentration and incubation time. The modifications have been interpreted as mainly due to insertion of nifedipine into the outer layer of the RBC membrane. The potential effect of nifedipine as a hemoglobin denaturant has been ruled out by x-ray absorption near-edge structure and optical spectroscopies.

Thermal analysis and microstructural characterization of Mg-Al-Zn system alloys

NASA Astrophysics Data System (ADS)

Król, M.; Tański, T.; Sitek, W.

2015-11-01

The influence of Zn amount and solidification rate on the characteristic temperature of the evaluation of magnesium dendrites during solidification at different cooling rates (0.6-2.5°C) were examined by thermal derivative analysis (TDA). The dendrite coherency point (DCP) is presented with a novel approach based on second derivative cooling curve. Solidification behavior was examined via one thermocouple thermal analysis method. Microstructural assessments were described by optical light microscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy. These studies showed that utilization of d2T/dt2 vs. the time curve methodology provides for analysis of the dendrite coherency point

Light sheet-based fluorescence microscopy (LSFM) reduces phototoxic effects and provides new means for the modern life sciences

NASA Astrophysics Data System (ADS)

Pampaloni, Francesco; Ansari, Nari; Girard, Philippe; Stelzer, Ernst H. K.

2011-07-01

Most optical technologies are applied to flat, basically two-dimensional cellular systems. However, physiological meaningful information relies on the morphology, the mechanical properties and the biochemistry of a cell's context. A cell requires the complex three-dimensional relationship to other cells. However, the observation of multi-cellular biological specimens remains a challenge. Specimens scatter and absorb light, thus, the delivery of the probing light and the collection of the signal light become inefficient; many endogenous biochemical compounds also absorb light and suffer degradation of some sort (photo-toxicity), which induces malfunction of a specimen. In conventional and confocal fluorescence microscopy, whenever a single plane, the entire specimen is illuminated. Recording stacks of images along the optical Z-axis thus illuminates the entire specimen once for each plane. Hence, cells are illuminated 10-20 and fish 100-300 times more often than they are observed. This can be avoided by changing the optical arrangement. The basic idea is to use light sheets, which are fed into the specimen from the side and overlap with the focal plane of a wide-field fluorescence microscope. In contrast to an epi-fluorescence arrangement, such an azimuthal fluorescence arrangement uses two independently operated lenses for illumination and detection. Optical sectioning and no photo-toxic damage or photo-bleaching outside a small volume close to the focal plane are intrinsic properties. Light sheet-based fluorescence microscopy (LSFM) takes advantage of modern camera technologies. LSFM can be operated with laser cutters and for fluorescence correlation spectroscopy. During the last few years, LSFM was used to record zebrafish development from the early 32-cell stage until late neurulation with sub-cellular resolution and short sampling periods (60-90 sec/stack). The recording speed was five 4-Megapixel large frames/sec with a dynamic range of 12-14 bit. We followed cell movements during gastrulation, revealed the development during cell migration processes and showed that an LSFM exposes an embryo to 200 times less energy than a conventional and 5,000 times less energy than a confocal fluorescence microscope. Most recently, we implemented incoherent structured illumination in our DSLM. The intensity modulated light sheets can be generated with dynamic frequencies and allow us to estimate the effect of the specimen on the image formation process at various depths in objects of different age.

Slide-free histology via MUSE: UV surface excitation microscopy for imaging unsectioned tissue (Conference Presentation)

NASA Astrophysics Data System (ADS)

Levenson, Richard M.; Harmany, Zachary; Demos, Stavros G.; Fereidouni, Farzad

2016-03-01

Widely used methods for preparing and viewing tissue specimens at microscopic resolution have not changed for over a century. They provide high-quality images but can involve time-frames of hours or even weeks, depending on logistics. There is increasing interest in slide-free methods for rapid tissue analysis that can both decrease turn-around times and reduce costs. One new approach is MUSE (microscopy with UV surface excitation), which exploits the shallow penetration of UV light to excite fluorescent signals from only the most superficial tissue elements. The method is non-destructive, and eliminates requirement for conventional histology processing, formalin fixation, paraffin embedding, or thin sectioning. It requires no lasers, confocal, multiphoton or optical coherence tomography optics. MUSE generates diagnostic-quality histological images that can be rendered to resemble conventional hematoxylin- and eosin-stained samples, with enhanced topographical information, from fresh or fixed, but unsectioned tissue, rapidly, with high resolution, simply and inexpensively. We anticipate that there could be widespread adoption in research facilities, hospital-based and stand-alone clinical settings, in local or regional pathology labs, as well as in low-resource environments.

Simultaneous confocal fluorescence microscopy and optical coherence tomography for drug distribution and tissue integrity assessment

NASA Astrophysics Data System (ADS)

Rinehart, Matthew T.; LaCroix, Jeffrey; Henderson, Marcus; Katz, David; Wax, Adam

2011-03-01

The effectiveness of microbicidal gels, topical products developed to prevent infection by sexually transmitted diseases including HIV/AIDS, is governed by extent of gel coverage, pharmacokinetics of active pharmaceutical ingredients (APIs), and integrity of vaginal epithelium. While biopsies provide localized information about drug delivery and tissue structure, in vivo measurements are preferable in providing objective data on API and gel coating distribution as well as tissue integrity. We are developing a system combining confocal fluorescence microscopy with optical coherence tomography (OCT) to simultaneously measure local concentrations and diffusion coefficients of APIs during transport from microbicidal gels into tissue, while assessing tissue integrity. The confocal module acquires 2-D images of fluorescent APIs multiple times per second allowing analysis of lateral diffusion kinetics. The custom Fourier domain OCT module has a maximum a-scan rate of 54 kHz and provides depth-resolved tissue integrity information coregistered with the confocal fluorescence measurements. The combined system is validated by imaging phantoms with a surrogate fluorophore. Time-resolved API concentration measured at fixed depths is analyzed for diffusion kinetics. This multimodal system will eventually be implemented in vivo for objective evaluation of microbicide product performance.

Controlling the influence of elastic eigenmodes on nanomagnet dynamics through pattern geometry

NASA Astrophysics Data System (ADS)

Berk, C.; Yahagi, Y.; Dhuey, S.; Cabrini, S.; Schmidt, H.

2017-03-01

The effect of the nanoscale array geometry on the interaction between optically generated surface acoustic waves (SAWs) and nanomagnet dynamics is investigated using Time-Resolved Magneto-Optical Kerr Effect Microscopy (TR-MOKE). It is demonstrated that altering the nanomagnet geometry from a periodic to a randomized aperiodic pattern effectively removes the magneto-elastic effect of SAWs on the magnetization dynamics. The efficiency of this method depends on the extent of any residual spatial correlations and is quantified by spatial Fourier analysis of the two structures. Randomization allows observation and extraction of intrinsic magnetic parameters such as spin wave frequencies and damping to be resolvable using all-optical methods, enabling the conclusion that the fabrication process does not affect the damping.

Optical Diagnostics in Medicine

NASA Astrophysics Data System (ADS)

Iftimia, Nicusor

2003-03-01

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

Real-time mapping of salt glands on the leaf surface of Cynodon dactylon L. using scanning electrochemical microscopy.

PubMed

Parthasarathy, Meera; Pemaiah, Brindha; Natesan, Ravichandran; Padmavathy, Saralla R; Pachiappan, Jayaraman

2015-02-01

Salt glands are specialized organelles present in the leaf tissues of halophytes, which impart salt-tolerance capability to the plant species. These glands are usually identified only by their morphology using conventional staining procedures coupled with optical microscopy. In this work, we have employed scanning electrochemical microscopy to identify the salt glands not only by their morphology but also by their salt excretion behavior. Bermuda grass (Cynodon dactylon L.) species was chosen for the study as they are known to be salt-tolerant and contain salt glands on leaf surfaces. Scanning electrochemical microscopy performed in sodium chloride medium in the presence and absence of potassium ferrocyanide as redox mediator, reveals the identity of salt glands. More insight into the ion expulsion behavior of these glands was obtained by mapping lateral and vertical variations in ion concentrations using surface impedance measurements which indicated five times higher resistance over the salt glands compared to the surrounding tissues and bulk solution. The protocol could be used to understand the developmental processes in plants grown in different soil/water conditions in order to improve salt tolerance of food crops by genetic engineering and hence improve their agricultural productivity.

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

PubMed

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

2009-10-26

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

A versatile soft X-ray transmission system for time resolved in situ microscopy with chemical contrast.

PubMed

Forsberg, J; Englund, C-J; Duda, L-C

2009-08-01

We present the design and operation of a versatile soft X-ray transmission system for time resolved in situ microscopy with chemical contrast. The utility of the setup is demonstrated by results from following a corrosion process of iron in saline environment, subjected to a controlled humid atmosphere. The system includes a transmission flow-cell reactor that allows for in situ microscopic probing with soft X-rays. We employ a full field technique by using a nearly collimated X-ray beam that produces an unmagnified projection of the transmitted soft X-rays (below 1.1 keV) which is magnified and recorded by an optical CCD camera. Time lapse series with chemical contrast allow us to follow and interpret the chemical processes in detail. The obtainable lateral resolution is a few mum, sufficient to detect filiform corrosion on iron.

CXRO - Mi-Young Im, Staff Scientist

Science.gov Websites

X-Ray Database Zone Plate Education Nanomagnetism X-Ray Microscopy LDJIM EUV Lithography EUV Mask Publications Contact The Center for X-Ray Optics is a multi-disciplined research group within Lawrence Berkeley -Ray Optics X-Ray Database Nanomagnetism X-Ray Microscopy EUV Lithography EUV Mask Imaging

Multifocal Fluorescence Microscope for Fast Optical Recordings of Neuronal Action Potentials

PubMed Central

Shtrahman, Matthew; Aharoni, Daniel B.; Hardy, Nicholas F.; Buonomano, Dean V.; Arisaka, Katsushi; Otis, Thomas S.

2015-01-01

In recent years, optical sensors for tracking neural activity have been developed and offer great utility. However, developing microscopy techniques that have several kHz bandwidth necessary to reliably capture optically reported action potentials (APs) at multiple locations in parallel remains a significant challenge. To our knowledge, we describe a novel microscope optimized to measure spatially distributed optical signals with submillisecond and near diffraction-limit resolution. Our design uses a spatial light modulator to generate patterned illumination to simultaneously excite multiple user-defined targets. A galvanometer driven mirror in the emission path streaks the fluorescence emanating from each excitation point during the camera exposure, using unused camera pixels to capture time varying fluorescence at rates that are ∼1000 times faster than the camera’s native frame rate. We demonstrate that this approach is capable of recording Ca2+ transients resulting from APs in neurons labeled with the Ca2+ sensor Oregon Green Bapta-1 (OGB-1), and can localize the timing of these events with millisecond resolution. Furthermore, optically reported APs can be detected with the voltage sensitive dye DiO-DPA in multiple locations within a neuron with a signal/noise ratio up to ∼40, resolving delays in arrival time along dendrites. Thus, the microscope provides a powerful tool for photometric measurements of dynamics requiring submillisecond sampling at multiple locations. PMID:25650920

In situ microscopy for online monitoring of cell concentration in Pichia pastoris cultivations.

PubMed

Marquard, D; Enders, A; Roth, G; Rinas, U; Scheper, T; Lindner, P

2016-09-20

In situ Microscopy (ISM) is an optical non-invasive technique to monitor cells in bioprocesses in real-time. Pichia pastoris is one of the most promising protein expression systems. This yeast combines fast growth on simple media and important eukaryotic features such as glycosylation. In this work, the ISM technology was applied to Pichia pastoris cultivations for online monitoring of the cell concentration during cultivation. Different ISM settings were tested. The acquired images were analyzed with two image processing algorithms. In seven cultivations the cell concentration was monitored by the applied algorithms and offline samples were taken to determine optical density (OD) and dry cell mass (DCM). Cell concentrations up to 74g/L dry cell mass could be analyzed via the ISM. Depending on the algorithm and the ISM settings, an accuracy between 0.3 % and 12 % was achieved. The overall results show that for a robust measurement a combination of the two described algorithms is required. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantitative phase microscopy via optimized inversion of the phase optical transfer function.

PubMed

Jenkins, Micah H; Gaylord, Thomas K

2015-10-01

Although the field of quantitative phase imaging (QPI) has wide-ranging biomedical applicability, many QPI methods are not well-suited for such applications due to their reliance on coherent illumination and specialized hardware. By contrast, methods utilizing partially coherent illumination have the potential to promote the widespread adoption of QPI due to their compatibility with microscopy, which is ubiquitous in the biomedical community. Described herein is a new defocus-based reconstruction method that utilizes a small number of efficiently sampled micrographs to optimally invert the partially coherent phase optical transfer function under assumptions of weak absorption and slowly varying phase. Simulation results are provided that compare the performance of this method with similar algorithms and demonstrate compatibility with large phase objects. The accuracy of the method is validated experimentally using a microlens array as a test phase object. Lastly, time-lapse images of live adherent cells are obtained with an off-the-shelf microscope, thus demonstrating the new method's potential for extending QPI capability widely in the biomedical community.

The influence of surface microstructure and chemical composition on corrosion behaviour in fuel-grade bio-ethanol of low-alloy steel modified by plasma nitro-carburizing and post-oxidizing

NASA Astrophysics Data System (ADS)

Boniatti, Rosiana; Bandeira, Aline L.; Crespi, Ângela E.; Aguzzoli, Cesar; Baumvol, Israel J. R.; Figueroa, Carlos A.

2013-09-01

The interaction of bio-ethanol on steel surfaces modified by plasma-assisted diffusion technologies is studied for the first time. The influence of surface microstructure and chemical composition on corrosion behaviour of AISI 4140 low-alloy steel in fuel-grade bio-ethanol was investigated. The steel surfaces were modified by plasma nitro-carburizing followed plasma oxidizing. X-ray diffraction, scanning electron microscopy, optical microscopy, X-ray dispersive spectroscopy, and glow-discharge optical emission spectroscopy were used to characterize the modified surface before and after immersion tests in bio-ethanol up to 77 days. The main corrosion mechanism is pit formation. The pit density and pit size were measured in order to quantify the corrosion resistance which was found to depend more strongly on microstructure and morphology of the oxide layer than on its thickness. The best corrosion protection was observed for samples post-oxidized at 480 °C and 90 min.

Monitoring the process of pulmonary melanoma metastasis using large area and label-free nonlinear optical microscopy

NASA Astrophysics Data System (ADS)

Hua, Daozhu; Qi, Shuhong; Li, Hui; Zhang, Zhihong; Fu, Ling

2012-06-01

We performed large area nonlinear optical microscopy (NOM) for label-free monitoring of the process of pulmonary melanoma metastasis ex vivo with subcellular resolution in C57BL/6 mice. Multiphoton autofluorescence (MAF) and second harmonic generation (SHG) images of lung tissue are obtained in a volume of ~2.2 mm×2.2 mm×30 μm. Qualitative differences in morphologic features and quantitative measurement of pathological lung tissues at different time points are characterized. We find that combined with morphological features, the quantitative parameters, such as the intensity ratio of MAF and SHG between pathological tissue and normal tissue and the MAF to SHG index versus depth clearly shows the tissue physiological changes during the process of pulmonary melanoma metastasis. Our results demonstrate that large area NOM succeeds in monitoring the process of pulmonary melanoma metastasis, which can provide a powerful tool for the research in tumor pathophysiology and therapy evaluation.

Cellular Oxygen and Nutrient Sensing in Microgravity Using Time-Resolved Fluorescence Microscopy

NASA Technical Reports Server (NTRS)

Szmacinski, Henryk

2003-01-01

Oxygen and nutrient sensing is fundamental to the understanding of cell growth and metabolism. This requires identification of optical probes and suitable detection technology without complex calibration procedures. Under this project Microcosm developed an experimental technique that allows for simultaneous imaging of intra- and inter-cellular events. The technique consists of frequency-domain Fluorescence Lifetime Imaging Microscopy (FLIM), a set of identified oxygen and pH probes, and methods for fabrication of microsensors. Specifications for electronic and optical components of FLIM instrumentation are provided. Hardware and software were developed for data acquisition and analysis. Principles, procedures, and representative images are demonstrated. Suitable lifetime sensitive oxygen, pH, and glucose probes for intra- and extra-cellular measurements of analyte concentrations have been identified and tested. Lifetime sensing and imaging have been performed using PBS buffer, culture media, and yeast cells as a model systems. Spectral specifications, calibration curves, and probes availability are also provided in the report.

Synthesis and characterization of thermally evaporated Cu2SnSe3 ternary semiconductor

NASA Astrophysics Data System (ADS)

Hamdani, K.; Chaouche, M.; Benabdeslem, M.; Bechiri, L.; Benslim, N.; Amara, A.; Portier, X.; Bououdina, M.; Otmani, A.; Marie, P.

2014-11-01

Copper Tin Selenide (CuSnSe) powder was mechanically alloyed by high energy planetary ball milling, starting from elemental powders. Synthesis time and velocity have been optimized to produce Cu2SnSe3 materials. Thin films were prepared by thermal evaporation on Corning glass substrate at Ts = 300 °C. The structural, compositional, morphological and optical properties of the synthesized semiconductor have been analyzed by X-ray diffraction (XRD), energy dispersive X-ray analysis (EDAX), scanning electron microscopy (SEM) and transmission electron microscopy. The analyzed powder exhibited a cubic crystal structure, with the presence of Cu2Se as a secondary phase. On the other hand, the deposited films showed a cubic Cu2SnSe3 ternary phase and extra peaks belonging to some binary compounds. Furthermore, optical measurements showed that the deposited layers have a relatively high absorption coefficient of 105 cm-1 and present a band gap of 0.94 eV.

Scene-based Shack-Hartmann wavefront sensor for light-sheet microscopy

NASA Astrophysics Data System (ADS)

Lawrence, Keelan; Liu, Yang; Dale, Savannah; Ball, Rebecca; VanLeuven, Ariel J.; Sornborger, Andrew; Lauderdale, James D.; Kner, Peter

2018-02-01

Light-sheet microscopy is an ideal imaging modality for long-term live imaging in model organisms. However, significant optical aberrations can be present when imaging into an organism that is hundreds of microns or greater in size. To measure and correct optical aberrations, an adaptive optics system must be incorporated into the microscope. Many biological samples lack point sources that can be used as guide stars with conventional Shack-Hartmann wavefront sensors. We have developed a scene-based Shack-Hartmann wavefront sensor for measuring the optical aberrations in a light-sheet microscopy system that does not require a point-source and can measure the aberrations for different parts of the image. The sensor has 280 lenslets inside the pupil, creates an image from each lenslet with a 500 micron field of view and a resolution of 8 microns, and has a resolution for the wavefront gradient of 75 milliradians per lenslet. We demonstrate the system on both fluorescent bead samples and zebrafish embryos.

Single-spin stochastic optical reconstruction microscopy

PubMed Central

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

2014-01-01

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

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.

Optimization of Electrical Methods for Sub -surface Monitoring of Biological Contamination: From Micro-scale to Macroscopic one through Sub-micrometric Topographic and Electrochemical Studies of Oxydation/Reduction Processes Provoked by Bacteria

NASA Astrophysics Data System (ADS)

Dhahri, S.; Marliere, C.

2012-12-01

The presence of biological matter (bacteria) in deep geological sites for storage of, for instance, radioactive elements or groundwater in aquifers was clearly proved. That biomass triggers physical and chemical processes which greatly modify the durability and the sustainability of the storage sites. These processes, mainly from oxidative/reductive reactions, are poorly understood. This is mainly due to the fact that former studies were done at the macroscopic level far away from the micrometric scale where relevant processes induced by bacteria take place. Investigations at microscopic level are needed. Thus, we developed an experimental set -up based on the combined use of optical microscopy (epifluorescence and transmission), atomic force microscopy (AFM) and scanning electro -chemical microscopy (SECM) in order to get simultaneous information on topographic and electro -chemical processes at different length scales. The first highly sensitive step was to use AFM and optical microscopy with biological samples in liquid environment: We will present a new, non -perturbative method for imaging bacteria in their natural liquid environment using AFM. No immobilization protocol, neither chemical nor mechanical, is needed, contrary to what has been regarded till now as essential. Furthermore we were able to follow the natural gliding movements of bacteria, directly proving their living state during the AFM investigation: we thus directly prove the low impact of these breakthrough AFM observations on the native behavior of the bacteria. The second delicate step was to combine AFM and optical measurements with electrical ones. We mounted a new experimental set-up coupling real -time (i) monitoring of optical properties as the optical density (OD) evolution related to bulk bacterial growth in liquid or as the counting of number of bacteria adhering on the surface of the sample as well and (ii) electrical and electrochemical measurements. We thus will present results on the observed crossed correlations between physical, chemical and biological processes induced by the studied bacteria and the resulting variations of electrical signals as measured at different length scales. We indeed used variable sizes for the electrodes - from 10cm -square (colonies of around 10000 bacteria) to 0.1-1microns -square (the scale of an individual cell) thanks to newly manufactured AFM -SECM probes (using Focused Ion Beam - FIB method). These experiments were done with several bacterial strains, various medias (inoculated by bacteria versus non -inoculated). Furthermore, these results will shortly be applied to the optimized monitoring of the in -situ activity of bacteria consuming oil pollutants, following this way, in real -time, the bioremediation of an oil -contaminated soil (ANR ECOTECH_BIOPHY program).

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

NASA Astrophysics Data System (ADS)

Ballard, Stephen G.

1990-08-01

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

The Scanning Optical Microscope: An Overview

NASA Astrophysics Data System (ADS)

Kino, G. S.; Corte, T. R.; Xiao, G. Q.

1988-07-01

In the last few years there has been a resurgence in research on optical microscopes. One reason stems from the invention of the acoustic microscope by Quate and Lemons,1 and the realization that some of the same principles could be applied to the optical microscope. The acoustic microscope has better transverse definition for the same wavelength than the standard optical microscope and at the same time has far better range definition. Consequently, Kompfner, who was involved with the work on the early acoustic microscope, decided to try out similar scanning microscope principles with optics, and started a group with Wilson and Sheppard to carry out such research at Oxford.2 Sometime earlier, Petran et a13 had invented the tandem scanning microscope which used many of the same principles. Now, in our laboratory at Stanford, these ideas on the tandem scanning microscope and the scanning optical microscope are converging. Another aspect of this work, which stems from the earlier experience with the acoustic microscope, involves measurement of both phase and amplitude of the optical beam. It is also possible to use scanned optical microscopy for other purposes. For instance, an optical beam can be used to excite electrons and holes in semiconductors, and the generated current can be measured. By scanning the optical beam over the semiconductor, an image can be obtained of the regions where there is strong or weak electron hole generation. This type of microscope is called OBIC (Optical Beam Induced Current). A second application involves fluorescent imaging of biological materials. Here we have the excellent range definition of a scanning optical microscope which eliminates unwanted glare from regions of the material where the beam is unfocused.3 A third application is focused on the heating effect of the light beam. With such a system, images can be obtained which are associated with changes in the thermal properties of a material, changes in recombination rates in semiconductors, and differences in material properties associated with either acoustic or thermal effects.4,5 Thus, the range of scanning optical microscopy applications is very large. In the main, the most important applications have been to semiconductors and to biology.

Handheld Fluorescence Microscopy based Flow Analyzer.

PubMed

Saxena, Manish; Jayakumar, Nitin; Gorthi, Sai Siva

2016-03-01

Fluorescence microscopy has the intrinsic advantages of favourable contrast characteristics and high degree of specificity. Consequently, it has been a mainstay in modern biological inquiry and clinical diagnostics. Despite its reliable nature, fluorescence based clinical microscopy and diagnostics is a manual, labour intensive and time consuming procedure. The article outlines a cost-effective, high throughput alternative to conventional fluorescence imaging techniques. With system level integration of custom-designed microfluidics and optics, we demonstrate fluorescence microscopy based imaging flow analyzer. Using this system we have imaged more than 2900 FITC labeled fluorescent beads per minute. This demonstrates high-throughput characteristics of our flow analyzer in comparison to conventional fluorescence microscopy. The issue of motion blur at high flow rates limits the achievable throughput in image based flow analyzers. Here we address the issue by computationally deblurring the images and show that this restores the morphological features otherwise affected by motion blur. By further optimizing concentration of the sample solution and flow speeds, along with imaging multiple channels simultaneously, the system is capable of providing throughput of about 480 beads per second.

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).

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

PubMed

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

2015-03-01

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

Computational adaptive optics for broadband optical interferometric tomography of biological tissue.

PubMed

Adie, Steven G; Graf, Benedikt W; Ahmad, Adeel; Carney, P Scott; Boppart, Stephen A

2012-05-08

Aberrations in optical microscopy reduce image resolution and contrast, and can limit imaging depth when focusing into biological samples. Static correction of aberrations may be achieved through appropriate lens design, but this approach does not offer the flexibility of simultaneously correcting aberrations for all imaging depths, nor the adaptability to correct for sample-specific aberrations for high-quality tomographic optical imaging. Incorporation of adaptive optics (AO) methods have demonstrated considerable improvement in optical image contrast and resolution in noninterferometric microscopy techniques, as well as in optical coherence tomography. Here we present a method to correct aberrations in a tomogram rather than the beam of a broadband optical interferometry system. Based on Fourier optics principles, we correct aberrations of a virtual pupil using Zernike polynomials. When used in conjunction with the computed imaging method interferometric synthetic aperture microscopy, this computational AO enables object reconstruction (within the single scattering limit) with ideal focal-plane resolution at all depths. Tomographic reconstructions of tissue phantoms containing subresolution titanium-dioxide particles and of ex vivo rat lung tissue demonstrate aberration correction in datasets acquired with a highly astigmatic illumination beam. These results also demonstrate that imaging with an aberrated astigmatic beam provides the advantage of a more uniform depth-dependent signal compared to imaging with a standard gaussian beam. With further work, computational AO could enable the replacement of complicated and expensive optical hardware components with algorithms implemented on a standard desktop computer, making high-resolution 3D interferometric tomography accessible to a wider group of users and nonspecialists.

Fabrication and characterization of a nanometer-sized optical fiber electrode based on selective chemical etching for scanning electrochemical/optical microscopy.

PubMed

Maruyama, Kenichi; Ohkawa, Hiroyuki; Ogawa, Sho; Ueda, Akio; Niwa, Osamu; Suzuki, Koji

2006-03-15

We have already reported a method for fabricating ultramicroelectrodes (Suzuki, K. JP Patent, 2004-45394, 2004). This method is based on the selective chemical etching of optical fibers. In this work, we undertake a detailed investigation involving a combination of etched optical fibers with various types of tapered tip (protruding-shape, double- (or pencil-) shape and triple-tapered electrode) and insulation with electrophoretic paint. Our goal is to establish a method for fabricating nanometer-sized optical fiber electrodes with high reproducibility. As a result, we realized pencil-shaped and triple-tapered electrodes that had radii in the nanometer range with high reproducibility. These nanometer-sized electrodes showed well-defined sigmoidal curves and stable diffusion-limited responses with cyclic voltammetry. The pencil-shaped optical fiber, which has a conical tip with a cone angle of 20 degrees , was effective for controlling the electrode radius. The pencil-shaped electrodes had higher reproducibility and smaller electrode radii (r(app) < 1.0 nm) than those of other etched optical fiber electrodes. By using a pencil-shaped electrode with a 105-nm radius as a probe, we obtained simultaneous electrochemical and optical images of an implantable interdigitated array electrode. We achieved nanometer-scale resolution with a combination of scanning electrochemical microscopy SECM and optical microscopy. The resolution of the electrochemical and optical images indicated sizes of 300 and 930 nm, respectively. The neurites of living PC12 cells were also successfully imaged on a 1.6-microm scale by using the negative feedback mode of an SECM.

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection

PubMed Central

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

2018-01-01

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

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

PubMed

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

2018-01-01

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

Sensitivity of photoacoustic microscopy

PubMed Central

Yao, Junjie; Wang, Lihong V.

2014-01-01

Building on its high spatial resolution, deep penetration depth and excellent image contrast, 3D photoacoustic microscopy (PAM) has grown tremendously since its first publication in 2005. Integrating optical excitation and acoustic detection, PAM has broken through both the optical diffusion and optical diffraction limits. PAM has 100% relative sensitivity to optical absorption (i.e., a given percentage change in the optical absorption coefficient yields the same percentage change in the photoacoustic amplitude), and its ultimate detection sensitivity is limited only by thermal noise. Focusing on the engineering aspects of PAM, this Review discusses the detection sensitivity of PAM, compares the detection efficiency of different PAM designs, and summarizes the imaging performance of various endogenous and exogenous contrast agents. It then describes representative PAM applications with high detection sensitivity, and outlines paths to further improvement. PMID:25302158

Ultrahigh-resolution optical coherence microscopy accurately classifies precancerous and cancerous human cervix free of labeling

PubMed Central

Zeng, Xianxu; Zhang, Xiaoan; Li, Canyu; Wang, Xiaofang; Jerwick, Jason; Xu, Tao; Ning, Yuan; Wang, Yihong; Zhang, Linlin; Zhang, Zhan; Ma, Yutao; Zhou, Chao

2018-01-01

Cervical cancer remains the fourth most common cause of cancer worldwide and the third leading cause of cancer deaths for women in developing countries. Traditional screening tools, such as human papillomavirus and Pap tests, cannot provide results in real-time and cannot localize suspicious regions. Colposcopy-directed biopsies are invasive in nature and only a few sites of the cervix may be chosen for investigation. A non-invasive, label-free and real-time imaging method with a resolution approaching that of histopathology is desirable for early detection of the disease. Methods: Ultrahigh-resolution optical coherence microscopy (OCM) is an emerging imaging technique used to obtain 3-dimensional (3-D) “optical biopsies” of biological samples with cellular resolution. In this study, 497 3-D OCM datasets from 159 specimens were collected from 92 patients. Results: Distinctive patterns for normal cervix, squamocolumnar junction, ectropion, low-grade and high-grade squamous intraepithelial lesions (LSIL and HSIL) and invasive cervical lesions were clearly observed from OCM images, which matched well with corresponding histological slides. OCM images demonstrated a sensitivity of 80% (95% confidence interval, CI, 72%-86%) and a specificity of 89% (95% CI, 84%-93%) for detecting high-risk lesions (HSIL and invasive lesions) when blindly tested by three investigators. A substantial inter-observer agreement was observed (κ=0.627), which showed high diagnostic consistency among three investigators. Conclusion: These results laid the foundation for future non-invasive optical evaluation of cervical tissue in vivo, which could lead to a less invasive and more effective screening and “see-and-treat” strategy for the management of cervical cancer. PMID:29896305

Neurite density from magnetic resonance diffusion measurements at ultrahigh field: Comparison with light microscopy and electron microscopy

PubMed Central

Jespersen, Sune N.; Bjarkam, Carsten R.; Nyengaard, Jens R.; Chakravarty, M. Mallar; Hansen, Brian; Vosegaard, Thomas; Østergaard, Leif; Yablonskiy, Dmitriy; Nielsen, Niels Chr.; Vestergaard-Poulsen, Peter

2010-01-01

Due to its unique sensitivity to tissue microstructure, diffusion-weighted magnetic resonance imaging (MRI) has found many applications in clinical and fundamental science. With few exceptions, a more precise correspondence between physiological or biophysical properties and the obtained diffusion parameters remain uncertain due to lack of specificity. In this work, we address this problem by comparing diffusion parameters of a recently introduced model for water diffusion in brain matter to light microscopy and quantitative electron microscopy. Specifically, we compare diffusion model predictions of neurite density in rats to optical myelin staining intensity and stereological estimation of neurite volume fraction using electron microscopy. We find that the diffusion model describes data better and that its parameters show stronger correlation with optical and electron microscopy, and thus reflect myelinated neurite density better than the more frequently used diffusion tensor imaging (DTI) and cumulant expansion methods. Furthermore, the estimated neurite orientations capture dendritic architecture more faithfully than DTI diffusion ellipsoids. PMID:19732836

In vivo correlation mapping microscopy

NASA Astrophysics Data System (ADS)

McGrath, James; Alexandrov, Sergey; Owens, Peter; Subhash, Hrebesh; Leahy, Martin

2016-04-01

To facilitate regular assessment of the microcirculation in vivo, noninvasive imaging techniques such as nailfold capillaroscopy are required in clinics. Recently, a correlation mapping technique has been applied to optical coherence tomography (OCT), which extends the capabilities of OCT to microcirculation morphology imaging. This technique, known as correlation mapping optical coherence tomography, has been shown to extract parameters, such as capillary density and vessel diameter, and key clinical markers associated with early changes in microvascular diseases. However, OCT has limited spatial resolution in both the transverse and depth directions. Here, we extend this correlation mapping technique to other microscopy modalities, including confocal microscopy, and take advantage of the higher spatial resolution offered by these modalities. The technique is achieved as a processing step on microscopy images and does not require any modification to the microscope hardware. Results are presented which show that this correlation mapping microscopy technique can extend the capabilities of conventional microscopy to enable mapping of vascular networks in vivo with high spatial resolution in both the transverse and depth directions.

Characterization of konjac glucomannan-ethyl cellulose film formation via microscopy.

PubMed

Xiao, Man; Wan, Li; Corke, Harold; Yan, Wenli; Ni, Xuewen; Fang, Yapeng; Jiang, Fatang

2016-04-01

Konjac glucomannan-ethyl cellulose (KGM-EC, 7:3, w/w) blended film shows good mechanical and moisture resistance properties. To better understand the basis for the KGM-EC film formation, optical microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) were used to observe the formation of the film from emulsion. Optical microscopy images showed that EC oil droplets were homogeneously dispersed in KGM water phase without obviously coalescence throughout the entire drying process. SEM images showed the surface and cross-sectional structures of samples maintained continuous and homogeneous appearance from the emulsion to dried film. AFM images indicated that KGM molecules entangled EC molecules in the emulsion. Interactions between KGM and EC improved the stability of KGM-EC emulsion, and contributed to uniformed structures of film formation. Based on these output information, a schematic model was built to elucidate KGM-EC film-forming process. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Image formation of volume holographic microscopy using point spread functions

NASA Astrophysics Data System (ADS)

Luo, Yuan; Oh, Se Baek; Kou, Shan Shan; Lee, Justin; Sheppard, Colin J. R.; Barbastathis, George

2010-04-01

We present a theoretical formulation to quantify the imaging properties of volume holographic microscopy (VHM). Volume holograms are formed by exposure of a photosensitive recording material to the interference of two mutually coherent optical fields. Recently, it has been shown that a volume holographic pupil has spatial and spectral sectioning capability for fluorescent samples. Here, we analyze the point spread function (PSF) to assess the imaging behavior of the VHM with a point source and detector. The coherent PSF of the VHM is derived, and the results are compared with those from conventional microscopy, and confocal microscopy with point and slit apertures. According to our analysis, the PSF of the VHM can be controlled in the lateral direction by adjusting the parameters of the VH. Compared with confocal microscopes, the performance of the VHM is comparable or even potentially better, and the VHM is also able to achieve real-time and three-dimensional (3D) imaging due to its multiplexing ability.

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

NASA Astrophysics Data System (ADS)

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

2017-08-01

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

Shaping field for deep tissue microscopy

NASA Astrophysics Data System (ADS)

Colon, J.; Lim, H.

2015-05-01

Information capacity of a lossless image-forming system is a conserved property determined by two imaging parameters - the resolution and the field of view (FOV). Adaptive optics improves the former by manipulating the phase, or wavefront, in the pupil plane. Here we describe a homologous approach, namely adaptive field microscopy, which aims to enhance the FOV by controlling the phase, or defocus, in the focal plane. In deep tissue imaging, the useful FOV can be severely limited if the region of interest is buried in a thick sample and not perpendicular to the optic axis. One must acquire many z-scans and reconstruct by post-processing, which exposes tissue to excessive radiation and is also time consuming. We demonstrate the effective FOV can be substantially enhanced by dynamic control of the image plane. Specifically, the tilt of the image plane is continuously adjusted in situ to match the oblique orientation of the sample plane within tissue. The utility of adaptive field microscopy is tested for imaging tissue with non-planar morphology. Ocular tissue of small animals was imaged by two-photon excited fluorescence. Our results show that adaptive field microscopy can utilize the full FOV. The freedom to adjust the image plane to account for the geometrical variations of sample could be extremely useful for 3D biological imaging. Furthermore, it could facilitate rapid surveillance of cellular features within deep tissue while avoiding photo damages, making it suitable for in vivo imaging.

Wide field fluorescence epi-microscopy behind a scattering medium enabled by speckle correlations

NASA Astrophysics Data System (ADS)

Hofer, Matthias; Soeller, Christian; Brasselet, Sophie; Bertolotti, Jacopo

2018-04-01

Fluorescence microscopy is widely used in biological imaging, however scattering from tissues strongly limits its applicability to a shallow depth. In this work we adapt a methodology inspired from stellar speckle interferometry, and exploit the optical memory effect to enable fluorescence microscopy through a turbid layer. We demonstrate efficient reconstruction of micrometer-size fluorescent objects behind a scattering medium in epi-microscopy, and study the specificities of this imaging modality (magnification, field of view, resolution) as compared to traditional microscopy. Using a modified phase retrieval algorithm to reconstruct fluorescent objects from speckle images, we demonstrate robust reconstructions even in relatively low signal to noise conditions. This modality is particularly appropriate for imaging in biological media, which are known to exhibit relatively large optical memory ranges compatible with tens of micrometers size field of views, and large spectral bandwidths compatible with emission fluorescence spectra of tens of nanometers widths.

Biomolecular Imaging with Coherent Nonlinear Vibrational Microscopy

PubMed Central

Chung, Chao-Yu; Boik, John; Potma, Eric O.

2014-01-01

Optical imaging with spectroscopic vibrational contrast is a label-free solution for visualizing, identifying, and quantifying a wide range of biomolecular compounds in biological materials. Both linear and nonlinear vibrational microscopy techniques derive their imaging contrast from infrared active or Raman allowed molecular transitions, which provide a rich palette for interrogating chemical and structural details of the sample. Yet nonlinear optical methods, which include both second-order sum-frequency generation (SFG) and third-order coherent Raman scattering (CRS) techniques, offer several improved imaging capabilities over their linear precursors. Nonlinear vibrational microscopy features unprecedented vibrational imaging speeds, provides strategies for higher spatial resolution, and gives access to additional molecular parameters. These advances have turned vibrational microscopy into a premier tool for chemically dissecting live cells and tissues. This review discusses the molecular contrast of SFG and CRS microscopy and highlights several of the advanced imaging capabilities that have impacted biological and biomedical research. PMID:23245525

Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection

PubMed Central

Zhi, Yanan; Wang, Benquan; Yao, Xincheng

2016-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2010-01-01

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

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.

Cytology 3D structure formation based on optical microscopy images

NASA Astrophysics Data System (ADS)

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

2017-01-01

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

Silicon technology-based micro-systems for atomic force microscopy/photon scanning tunnelling microscopy.

PubMed

Gall-Borrut, P; Belier, B; Falgayrettes, P; Castagne, M; Bergaud, C; Temple-Boyer, P

2001-04-01

We developed silicon nitride cantilevers integrating a probe tip and a wave guide that is prolonged on the silicon holder with one or two guides. A micro-system is bonded to a photodetector. The resulting hybrid system enables us to obtain simultaneously topographic and optical near-field images. Examples of images obtained on a longitudinal cross-section of an optical fibre are shown.

Microcontact Printing via a Polymer-Induced Liquid-Precursor (PILP) Process

DTIC Science & Technology

2002-04-01

applications that require high performance mechanical, electrical and/or optical properties resulting from controlled nano- and microstructural design...salts. The cover-slips were examined by optical microscopy, and then gold coated for scanning electron microscopy on a SEM JEOL JSM 6400 instrument [5...applications in the realm of biomimicry . Controlled growth of crystals with specific orientation can be achieved via the functional groups on the substrate

Orbital angular momentum light in microscopy

PubMed Central

2017-01-01

Light with a helical phase has had an impact on optical imaging, pushing the limits of resolution or sensitivity. Here, special emphasis will be given to classical light microscopy of phase samples and to Fourier filtering techniques with a helical phase profile, such as the spiral phase contrast technique in its many variants and areas of application. This article is part of the themed issue ‘Optical orbital angular momentum’. PMID:28069768

In-Fiber Magneto-Optic Devices Based on Ultrahigh Verdet Constant Organic Materials and Holey Fibers

DTIC Science & Technology

2009-02-02

protocols and a noise equivalent magnetic field sensitivity of ~ 100 pT/ VHz has been demonstrated. • Magneto-optic properties of magnetite - PMMA composite...nanoparticle - PMMA nanocomposite. We have used both transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) to...we expect to enhance it in our devices by their proper symmetrization as described above. Passive Poking core ^^ direction Magnetic AA

Optical Biomarkers of Serous and Mucinous Human Ovarian Tumor Assessed with Nonlinear Optics Microscopies

PubMed Central

Adur, Javier; Pelegati, Vitor B.; de Thomaz, Andre A.; Baratti, Mariana O.; Almeida, Diogo B.; Andrade, L. A. L. A.; Bottcher-Luiz, Fátima; Carvalho, Hernandes F.; Cesar, Carlos L.

2012-01-01

Background Nonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression. Methodology/Principal Findings We obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells. Conclusions/Significance NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions. PMID:23056557

Accurate prediction of collapse temperature using optical coherence tomography-based freeze-drying microscopy.

PubMed

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

2013-06-01

The objective of this study was to assess the feasibility of developing and applying a laboratory tool that can provide three-dimensional product structural information during freeze-drying and which can accurately characterize the collapse temperature (Tc ) of pharmaceutical formulations designed for freeze-drying. A single-vial freeze dryer coupled with optical coherence tomography freeze-drying microscopy (OCT-FDM) was developed to investigate the structure and Tc of formulations in pharmaceutically relevant products containers (i.e., freeze-drying in vials). OCT-FDM was used to measure the Tc and eutectic melt of three formulations in freeze-drying vials. The Tc as measured by OCT-FDM was found to be predictive of freeze-drying with a batch of vials in a conventional laboratory freeze dryer. The freeze-drying cycles developed using OCT-FDM data, as compared with traditional light transmission freeze-drying microscopy (LT-FDM), resulted in a significant reduction in primary drying time, which could result in a substantial reduction of manufacturing costs while maintaining product quality. OCT-FDM provides quantitative data to justify freeze-drying at temperatures higher than the Tc measured by LT-FDM and provides a reliable upper limit to setting a product temperature in primary drying. Copyright © 2013 Wiley Periodicals, Inc.

Polarization-modulated second harmonic generation ellipsometric microscopy at video rate.

PubMed

DeWalt, Emma L; Sullivan, Shane Z; Schmitt, Paul D; Muir, Ryan D; Simpson, Garth J

2014-08-19

Fast 8 MHz polarization modulation coupled with analytical modeling, fast beam-scanning, and synchronous digitization (SD) have enabled simultaneous nonlinear optical Stokes ellipsometry (NOSE) and polarized laser transmittance imaging with image acquisition rates up to video rate. In contrast to polarimetry, in which the polarization state of the exiting beam is recorded, NOSE enables recovery of the complex-valued Jones tensor of the sample that describes all polarization-dependent observables of the measurement. Every video-rate scan produces a set of 30 images (10 for each detector with three detectors operating in parallel), each of which corresponds to a different polarization-dependent result. Linear fitting of this image set contracts it down to a set of five parameters for each detector in second harmonic generation (SHG) and three parameters for the transmittance of the incident beam. These parameters can in turn be used to recover the Jones tensor elements of the sample. Following validation of the approach using z-cut quartz, NOSE microscopy was performed for microcrystals of both naproxen and glucose isomerase. When weighted by the measurement time, NOSE microscopy was found to provide a substantial (>7 decades) improvement in the signal-to-noise ratio relative to our previous measurements based on the rotation of optical elements and a 3-fold improvement relative to previous single-point NOSE approaches.

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

Fischer, Martin C., E-mail: Martin.Fischer@duke.edu; Wilson, Jesse W.; Robles, Francisco E.

Multiphoton microscopy has rapidly gained popularity in biomedical imaging and materials science because of its ability to provide three-dimensional images at high spatial and temporal resolution even in optically scattering environments. Currently the majority of commercial and home-built devices are based on two-photon fluorescence and harmonic generation contrast. These two contrast mechanisms are relatively easy to measure but can access only a limited range of endogenous targets. Recent developments in fast laser pulse generation, pulse shaping, and detection technology have made accessible a wide range of optical contrasts that utilize multiple pulses of different colors. Molecular excitation with multiple pulsesmore » offers a large number of adjustable parameters. For example, in two-pulse pump-probe microscopy, one can vary the wavelength of each excitation pulse, the detection wavelength, the timing between the excitation pulses, and the detection gating window after excitation. Such a large parameter space can provide much greater molecular specificity than existing single-color techniques and allow for structural and functional imaging without the need for exogenous dyes and labels, which might interfere with the system under study. In this review, we provide a tutorial overview, covering principles of pump-probe microscopy and experimental setup, challenges associated with signal detection and data processing, and an overview of applications.« less

Spatiotemporal polarization modulation microscopy with a microretarder array

NASA Astrophysics Data System (ADS)

Ding, Changqin; Ulcickas, James R. W.; Simpson, Garth J.

2018-02-01

A patterned microretarder array positioned in the rear conjugate plane of a microscope enables rapid polarizationdependent nonlinear optical microscopy. The pattern introduced to the array results in periodic modulation of the polarization-state of the incident light as a function of position within the field of view with no moving parts or active control. Introduction of a single stationary optical element and a fixed polarizer into the beam of a nonlinear optical microscope enabled nonlinear optical tensor recovery, which informs on local structure and orientation. Excellent agreement was observed between the measured and predicted second harmonic generation (SHG) of z-cut quartz, selected as a test system with well-established nonlinear optical properties. Subsequent studies of spatially varying samples further support the general applicability of this relatively simple strategy for detailed polarization analysis in both conventional and nonlinear optical imaging of structurally diverse samples.

Center for Adaptive Optics | Events

Science.gov Websites

Center for Adaptive Optics A University of California Science and Technology Center home 2015 AO Adaptive Optics and Wavefront Control in Microscopy and Ophthalmology Paris, France October 25-25 CfAO Adaptive Optics Institute for Scientist and Engineer Educators Members Calendar of Events Publications

Enhanced photo-response of porous silicon photo-detectors by embeddingTitanium-dioxide nano-particles

NASA Astrophysics Data System (ADS)

Ali, Hiba M.; Makki, Sameer A.; Abd, Ahmed N.

2018-05-01

Porous silicon (n-PS) films can be prepared by photoelectochemical etching (PECE) Silicon chips n - types with 15 (mA / cm2), in 15 minutes etching time on the fabrication nano-sized pore arrangement. By using X-ray diffraction measurement and atomic power microscopy characteristics (AFM), PS was investigated. It was also evaluated the crystallites size from (XRD) for the PS nanoscale. The atomic force microscopy confirmed the nano-metric size chemical fictionalization through the electrochemical etching that was shown on the PS surface chemical composition. The atomic power microscopy checks showed the roughness of the silicon surface. It is also notified (TiO2) preparation nano-particles that were prepared by pulse laser eradication in ethanol (PLAL) technique through irradiation with a Nd:YAG laser pulses TiO2 target that is sunk in methanol using 400 mJ of laser energy. It has been studied the structural, optical and morphological of TiO2NPs. It has been detected that through XRD measurement, (TiO2) NPs have been Tetragonal crystal structure. While with AFM measurements, it has been realized that the synthesized TiO2 particles are spherical with an average particle size in the (82 nm) range. It has been determined that the energy band gap of TiO2 NPs from optical properties and set to be in (5eV) range.The transmittance and reflectance spectra have determined the TiO2 NPs optical constants. It was reported the effectiveness of TiO2 NPs expansion on the PS Photodetector properties which exposes the benefits in (Al/PS/Si/Al). The built-in tension values depend on the etching time current density and laser flounce. Al/TiO2/PS/Si/Al photo-detector heterojunction have two response peaks that are situated at 350 nm and (700 -800nm) with max sensitivity ≈ 0.7 A/W. The maximum given detectivity is 9.38at ≈ 780 nm wavelength.

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

PubMed Central

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

2014-01-01

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

Hyperspectral imaging with laser-scanning sum-frequency generation microscopy

PubMed Central

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

2017-01-01

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

Optically coupled methods for microwave impedance microscopy

NASA Astrophysics Data System (ADS)

Johnston, Scott R.; Ma, Eric Yue; Shen, Zhi-Xun

2018-04-01

Scanning Microwave Impedance Microscopy (MIM) measurement of photoconductivity with 50 nm resolution is demonstrated using a modulated optical source. The use of a modulated source allows for the measurement of photoconductivity in a single scan without a reference region on the sample, as well as removing most topographical artifacts and enhancing signal to noise as compared with unmodulated measurement. A broadband light source with a tunable monochrometer is then used to measure energy resolved photoconductivity with the same methodology. Finally, a pulsed optical source is used to measure local photo-carrier lifetimes via MIM, using the same 50 nm resolution tip.

Development of Nomarski microscopy for quantitative determination of surface topography

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

Hartman, J. S.; Gordon, R. L.; Lessor, D. L.

1979-01-01

The use of Nomarski differential interference contrast (DIC) microscopy has been extended to provide nondestructive, quantitative analysis of a sample's surface topography. Theoretical modeling has determined the dependence of the image intensity on the microscope's optical components, the sample's optical properties, and the sample's surface orientation relative to the microscope. Results include expressions to allow the inversion of image intensity data to determine sample surface slopes. A commercial Nomarski system has been modified and characterized to allow the evaluation of the optical model. Data have been recorded with smooth, planar samples that verify the theoretical predictions.

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

PubMed

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

2017-06-28

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

Techniques for 3D tracking of single molecules with nanometer accuracy in living cells

NASA Astrophysics Data System (ADS)

Gardini, Lucia; Capitanio, Marco; Pavone, Francesco S.

2013-06-01

We describe a microscopy technique that, combining wide-field single molecule microscopy, bifocal imaging and Highly Inclined and Laminated Optical sheet (HILO) microscopy, allows a 3D tracking with nanometer accuracy of single fluorescent molecules in vitro and in living cells.