Correlative super-resolution fluorescence microscopy combined with optical coherence microscopy

NASA Astrophysics Data System (ADS)

Kim, Sungho; Kim, Gyeong Tae; Jang, Soohyun; Shim, Sang-Hee; Bae, Sung Chul

2015-03-01

Recent development of super-resolution fluorescence imaging technique such as stochastic optical reconstruction microscopy (STORM) and photoactived localization microscope (PALM) has brought us beyond the diffraction limits. It allows numerous opportunities in biology because vast amount of formerly obscured molecular structures, due to lack of spatial resolution, now can be directly observed. A drawback of fluorescence imaging, however, is that it lacks complete structural information. For this reason, we have developed a super-resolution multimodal imaging system based on STORM and full-field optical coherence microscopy (FF-OCM). FF-OCM is a type of interferometry systems based on a broadband light source and a bulk Michelson interferometer, which provides label-free and non-invasive visualization of biological samples. The integration between the two systems is simple because both systems use a wide-field illumination scheme and a conventional microscope. This combined imaging system gives us both functional information at a molecular level (~20nm) and structural information at the sub-cellular level (~1μm). For thick samples such as tissue slices, while FF-OCM is readily capable of imaging the 3D architecture, STORM suffer from aberrations and high background fluorescence that substantially degrade the resolution. In order to correct the aberrations in thick tissues, we employed an adaptive optics system in the detection path of the STORM microscope. We used our multimodal system to obtain images on brain tissue samples with structural and functional information.

Direct observation of single flexible polymers using single stranded DNA†

PubMed Central

Brockman, Christopher; Kim, Sun Ju

2012-01-01

Over the last 15 years, double stranded DNA (dsDNA) has been used as a model polymeric system for nearly all single polymer dynamics studies. However, dsDNA is a semiflexible polymer with markedly different molecular properties compared to flexible chains, including synthetic organic polymers. In this work, we report a new system for single polymer studies of flexible chains based on single stranded DNA (ssDNA). We developed a method to synthesize ssDNA for fluorescence microscopy based on rolling circle replication, which generates long strands (>65 kb) of ssDNA containing “designer” sequences, thereby preventing intramolecular base pair interactions. Polymers are synthesized to contain amine-modified bases randomly distributed along the backbone, which enables uniform labelling of polymer chains with a fluorescent dye to facilitate fluorescence microscopy and imaging. Using this approach, we synthesized ssDNA chains with long contour lengths (>30 μm) and relatively low dye loading ratios (~1 dye per 100 bases). In addition, we used epifluorescence microscopy to image single ssDNA polymer molecules stretching in flow in a microfluidic device. Overall, we anticipate that ssDNA will serve as a useful model system to probe the dynamics of polymeric materials at the molecular level. PMID:22956981

PScan 1.0: flexible software framework for polygon based multiphoton microscopy

NASA Astrophysics Data System (ADS)

Li, Yongxiao; Lee, Woei Ming

2016-12-01

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

Thin-film tunable filters for hyperspectral fluorescence microscopy

PubMed Central

Favreau, Peter; Hernandez, Clarissa; Lindsey, Ashley Stringfellow; Alvarez, Diego F.; Rich, Thomas; Prabhat, Prashant

2013-01-01

Abstract. Hyperspectral imaging is a powerful tool that acquires data from many spectral bands, forming a contiguous spectrum. Hyperspectral imaging was originally developed for remote sensing applications; however, hyperspectral techniques have since been applied to biological fluorescence imaging applications, such as fluorescence microscopy and small animal fluorescence imaging. The spectral filtering method largely determines the sensitivity and specificity of any hyperspectral imaging system. There are several types of spectral filtering hardware available for microscopy systems, most commonly acousto-optic tunable filters (AOTFs) and liquid crystal tunable filters (LCTFs). These filtering technologies have advantages and disadvantages. Here, we present a novel tunable filter for hyperspectral imaging—the thin-film tunable filter (TFTF). The TFTF presents several advantages over AOTFs and LCTFs, most notably, a high percentage transmission and a high out-of-band optical density (OD). We present a comparison of a TFTF-based hyperspectral microscopy system and a commercially available AOTF-based system. We have characterized the light transmission, wavelength calibration, and OD of both systems, and have then evaluated the capability of each system for discriminating between green fluorescent protein and highly autofluorescent lung tissue. Our results suggest that TFTFs are an alternative approach for hyperspectral filtering that offers improved transmission and out-of-band blocking. These characteristics make TFTFs well suited for other biomedical imaging devices, such as ophthalmoscopes or endoscopes. PMID:24077519

QUALITY ASSESSMENT OF CONFOCAL MICROSCOPY SLIDE-BASED SYSTEMS: INSTABLITY

EPA Science Inventory

Background: All slide-based fluorescence cytometry detections systems basically include an excitation light source, intermediate optics, and a detection device (CCD or PMT). Occasionally, this equipment becomes unstable, generating unreliable and inferior data. Methods: A num...

Intravital multiphoton fluorescence imaging and optical manipulation of spinal cord in mice, using a compact fiber laser system.

PubMed

Oshima, Yusuke; Horiuch, Hideki; Honkura, Naoki; Hikita, Atsuhiko; Ogata, Tadanori; Miura, Hiromasa; Imamura, Takeshi

2014-09-01

Near-infrared ultrafast lasers are widely used for multiphoton excited fluorescence microscopy in living animals. Ti:Sapphire lasers are typically used for multiphoton excitation, but their emission wavelength is restricted below 1,000 nm. The aim of this study is to evaluate the performance of a compact Ytterbium-(Yb-) fiber laser at 1,045 nm for multiphoton excited fluorescence microscopy in spinal cord injury. In this study, we employed a custom-designed microscopy system with a compact Yb-fiber laser and evaluated the performance of this system in in vivo imaging of brain cortex and spinal cord in YFP-H transgenic mice. For in vivo imaging of brain cortex, sharp images of basal dendrites, and pyramidal cells expressing EYFP were successfully captured using the Yb-fiber laser in our microscopy system. We also performed in vivo imaging of axon fibers of spinal cord in the transgenic mice. The obtained images were almost as sharp as those obtained using a conventional ultrafast laser system. In addition, laser ablation and multi-color imaging could be performed simultaneously using the Yb-fiber laser. The high-peak pulse Yb-fiber laser is potentially useful for multimodal bioimaging methods based on a multiphoton excited fluorescence microscopy system that incorporates laser ablation techniques. Our results suggest that microscopy systems of this type could be utilized in studies of neuroscience and clinical use in diagnostics and therapeutic tool for spinal cord injury in the future. © 2014 Wiley Periodicals, Inc.

Interfacial micromorphological differences in hybrid layer formation between water- and solvent-based dentin bonding systems.

PubMed

Gregoire, Geneviève L; Akon, Bernadette A; Millas, Arlette

2002-06-01

Many dentin bonding systems of different compositions, and in particular containing different solvents, have been introduced to the market. Their effect on the quality of the interface requires clarification by means of comparative trials. This study investigated micromorphological differences in hybrid layer formation with a variety of commercially available water- or solvent-based dentin bonding products and their recommended compomers. Five bonding systems were used on groups of 10 teeth each as follows: group I, acetone-based system used with 36% phosphoric acid; group II, a different acetone-based system containing nano-sized particles for filler loading and used with a non-rinsing conditioner containing maleic acid; group III, the acetone-based system of group II used with 36% phosphoric acid (the only difference in the treatment for groups II and III was the acid etching system); group IV, a mixed-solvent-based system (water/ethanol) used with 37% phosphoric acid; and group V, a water-based system used with 37% phosphoric acid. Each bonding system was covered with the recommended compomer. Class I occlusal preparations were made in extracted teeth and restored with one of the above systems. Five specimens of each group were studied with optical microscopy after staining. Scanning electron microscopy was used to examine the interface of the bonding system/dentin of the other 5 teeth in each group. The optical microscopy measurements were made with a 10 x 10 reticle. A micron mark with scale was used for the scanning electron microscope. All measurements were made in microm. The following criteria were used to define a good interface: absence of voids between the different parts of the interface, uniformity of the hybrid layer, good opening of the tubuli orifices, and tag adherence to the tubuli walls. Morphological differences were found at the interface depending on dentin treatment and adhesive composition. The acetone-containing systems were associated with a continuous, gap-free hybrid layer that was linked intimately with the dentin. The tags adhered well to the tubuli walls and were often joined by side branches. In the water-based solvent systems, a lack of contact was visible between the resin tags and the tubuli walls, with some incompletely filled tubuli and some gaps in the hybrid layer. The 2 observational methods used, optical and scanning electron microscopy, proved to be complementary. Within the limitations of this study, use of the acetone-based systems after phosphoric acid etching resulted in a continuous, thick hybrid layer with reverse-cone-shaped tags in close contact with the tubuli walls. Use of the water-based systems resulted in a thinner hybrid layer with some incompletely sealed dentinal tubules.

Development of a surface topography instrument for automotive textured steel plate

NASA Astrophysics Data System (ADS)

Wang, Zhen; Wang, Shenghuai; Chen, Yurong; Xie, Tiebang

2010-08-01

The surface topography of automotive steel plate is decisive to its stamping, painting and image clarity performances. For measuring this kind of surface topography, an instrument has been developed based on the principle of vertical scanning white light microscopy interference principle. The microscopy interference system of this instrument is designed based on the structure of Linnik interference microscopy. The 1D worktable of Z direction is designed and introduced in details. The work principle of this instrument is analyzed. In measuring process, the interference microscopy is derived as a whole and the measured surface is scanned in vertical direction. The measurement accuracy and validity is verified by templates. Surface topography of textured steel plate is also measured by this instrument.

Invited review article: Advanced light microscopy for biological space research.

PubMed

De Vos, Winnok H; Beghuin, Didier; Schwarz, Christian J; Jones, David B; van Loon, Jack J W A; Bereiter-Hahn, Juergen; Stelzer, Ernst H K

2014-10-01

As commercial space flights have become feasible and long-term extraterrestrial missions are planned, it is imperative that the impact of space travel and the space environment on human physiology be thoroughly characterized. Scrutinizing the effects of potentially detrimental factors such as ionizing radiation and microgravity at the cellular and tissue level demands adequate visualization technology. Advanced light microscopy (ALM) is the leading tool for non-destructive structural and functional investigation of static as well as dynamic biological systems. In recent years, technological developments and advances in photochemistry and genetic engineering have boosted all aspects of resolution, readout and throughput, rendering ALM ideally suited for biological space research. While various microscopy-based studies have addressed cellular response to space-related environmental stressors, biological endpoints have typically been determined only after the mission, leaving an experimental gap that is prone to bias results. An on-board, real-time microscopical monitoring device can bridge this gap. Breadboards and even fully operational microscope setups have been conceived, but they need to be rendered more compact and versatile. Most importantly, they must allow addressing the impact of gravity, or the lack thereof, on physiologically relevant biological systems in space and in ground-based simulations. In order to delineate the essential functionalities for such a system, we have reviewed the pending questions in space science, the relevant biological model systems, and the state-of-the art in ALM. Based on a rigorous trade-off, in which we recognize the relevance of multi-cellular systems and the cellular microenvironment, we propose a compact, but flexible concept for space-related cell biological research that is based on light sheet microscopy.

Invited Review Article: Advanced light microscopy for biological space research

NASA Astrophysics Data System (ADS)

De Vos, Winnok H.; Beghuin, Didier; Schwarz, Christian J.; Jones, David B.; van Loon, Jack J. W. A.; Bereiter-Hahn, Juergen; Stelzer, Ernst H. K.

2014-10-01

As commercial space flights have become feasible and long-term extraterrestrial missions are planned, it is imperative that the impact of space travel and the space environment on human physiology be thoroughly characterized. Scrutinizing the effects of potentially detrimental factors such as ionizing radiation and microgravity at the cellular and tissue level demands adequate visualization technology. Advanced light microscopy (ALM) is the leading tool for non-destructive structural and functional investigation of static as well as dynamic biological systems. In recent years, technological developments and advances in photochemistry and genetic engineering have boosted all aspects of resolution, readout and throughput, rendering ALM ideally suited for biological space research. While various microscopy-based studies have addressed cellular response to space-related environmental stressors, biological endpoints have typically been determined only after the mission, leaving an experimental gap that is prone to bias results. An on-board, real-time microscopical monitoring device can bridge this gap. Breadboards and even fully operational microscope setups have been conceived, but they need to be rendered more compact and versatile. Most importantly, they must allow addressing the impact of gravity, or the lack thereof, on physiologically relevant biological systems in space and in ground-based simulations. In order to delineate the essential functionalities for such a system, we have reviewed the pending questions in space science, the relevant biological model systems, and the state-of-the art in ALM. Based on a rigorous trade-off, in which we recognize the relevance of multi-cellular systems and the cellular microenvironment, we propose a compact, but flexible concept for space-related cell biological research that is based on light sheet microscopy.

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

Biological applications of phase-contrast electron microscopy.

PubMed

Nagayama, Kuniaki

2014-01-01

Here, I review the principles and applications of phase-contrast electron microscopy using phase plates. First, I develop the principle of phase contrast based on a minimal model of microscopy, introducing a double Fourier-transform process to mathematically formulate the image formation. Next, I explain four phase-contrast (PC) schemes, defocus PC, Zernike PC, Hilbert differential contrast, and schlieren optics, as image-filtering processes in the context of the minimal model, with particular emphases on the Zernike PC and corresponding Zernike phase plates. Finally, I review applications of Zernike PC cryo-electron microscopy to biological systems such as protein molecules, virus particles, and cells, including single-particle analysis to delineate three-dimensional (3D) structures of protein and virus particles and cryo-electron tomography to reconstruct 3D images of complex protein systems and cells.

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.

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.

Sub-100 nm resolution microscopy based on proximity projection grating scheme

PubMed Central

Hu, Feng; Somekh, Michael G.; Albutt, Darren J.; Webb, Kevin; Moradi, Emilia; See, Chung W.

2015-01-01

Structured illumination microscopy (SIM) has been widely used in life science imaging applications. The maximum resolution improvement of SIM, compared to conventional bright field system is a factor of 2. Here we present an approach to structured illumination microscopy using the proximity projection grating scheme (PPGS), which has the ability to further enhance the SIM resolution without invoking any nonlinearity response from the sample. With the PPGS-based SIM, sub-100 nm resolution has been obtained experimentally, and results corresponding to 2.4 times resolution improvement are presented. Furthermore, it will be shown that an improvement of greater than 3 times can be achieved. PMID:25715953

A series of fluorene-based two-photon absorbing molecules: synthesis, linear and nonlinear characterization, and bioimaging

PubMed Central

Andrade, Carolina D.; Yanez, Ciceron O.; Rodriguez, Luis; Belfield, Kevin D.

2010-01-01

The synthesis, structural, and photophysical characterization of a series of new fluorescent donor–acceptor and acceptor-acceptor molecules, based on the fluorenyl ring system, with two-photon absorbing properties is described. These new compounds exhibited large Stokes shifts, high fluorescent quantum yields, and, significantly, high two-photon absorption cross sections, making them well suited for two-photon fluorescence microscopy (2PFM) imaging. Confocal and two-photon fluorescence microscopy imaging of COS-7 and HCT 116 cells incubated with probe I showed endosomal selectivity, demonstrating the potential of this class of fluorescent probes in multiphoton fluorescence microscopy. PMID:20481596

Photonic crystal fibre enables short-wavelength two-photon laser scanning fluorescence microscopy with fura-2

NASA Astrophysics Data System (ADS)

McConnell, Gail; Riis, Erling

2004-10-01

We report on a novel and compact reliable laser source capable of short-wavelength two-photon laser scanning fluorescence microscopy based on soliton self-frequency shift effects in photonic crystal fibre. We demonstrate the function of the system by performing two-photon microscopy of smooth muscle cells and cardiac myocytes from the rat pulmonary vein and Chinese hamster ovary cells loaded with the fluorescent calcium indicator fura-2/AM.

Microscopic video observation of capillary vessel systems using diffuse back lighting

NASA Astrophysics Data System (ADS)

Sakai, Minako; Arai, Hiroki; Iwai, Toshiaki

2017-04-01

We have been developing a simple and practical video microscopy system based on absorption spectra of biological substance to perform spectroscopic observation of living tissues. The diffuse backlighting effect is actively used in the developed system, which is generated by multiple light scattering in the tissue. It is demonstrated that the light specularly reflected from the skin surface can be completely suppressed in the microscopic observation and the biological activity of the capillary vessel systems distributed under the skin can be successfully observed. As a result, we can confirm the effectiveness of the video microscopy system using diffuse backlighting and the applicability of our developed system.

Going glass to digital: virtual microscopy as a simulation-based revolution in pathology and laboratory science.

PubMed

Nelson, Danielle; Ziv, Amitai; Bandali, Karim S

2012-10-01

The recent technological advance of digital high resolution imaging has allowed the field of pathology and medical laboratory science to undergo a dramatic transformation with the incorporation of virtual microscopy as a simulation-based educational and diagnostic tool. This transformation has correlated with an overall increase in the use of simulation in medicine in an effort to address dwindling clinical resource availability and patient safety issues currently facing the modern healthcare system. Virtual microscopy represents one such simulation-based technology that has the potential to enhance student learning and readiness to practice while revolutionising the ability to clinically diagnose pathology collaboratively across the world. While understanding that a substantial amount of literature already exists on virtual microscopy, much more research is still required to elucidate the full capabilities of this technology. This review explores the use of virtual microscopy in medical education and disease diagnosis with a unique focus on key requirements needed to take this technology to the next level in its use in medical education and clinical practice.

Republished: going glass to digital: virtual microscopy as a simulation-based revolution in pathology and laboratory science.

PubMed

Nelson, Danielle; Ziv, Amitai; Bandali, Karim S

2013-10-01

The recent technological advance of digital high resolution imaging has allowed the field of pathology and medical laboratory science to undergo a dramatic transformation with the incorporation of virtual microscopy as a simulation-based educational and diagnostic tool. This transformation has correlated with an overall increase in the use of simulation in medicine in an effort to address dwindling clinical resource availability and patient safety issues currently facing the modern healthcare system. Virtual microscopy represents one such simulation-based technology that has the potential to enhance student learning and readiness to practice while revolutionising the ability to clinically diagnose pathology collaboratively across the world. While understanding that a substantial amount of literature already exists on virtual microscopy, much more research is still required to elucidate the full capabilities of this technology. This review explores the use of virtual microscopy in medical education and disease diagnosis with a unique focus on key requirements needed to take this technology to the next level in its use in medical education and clinical practice.

Model-free iterative control of repetitive dynamics for high-speed scanning in atomic force microscopy.

PubMed

Li, Yang; Bechhoefer, John

2009-01-01

We introduce an algorithm for calculating, offline or in real time and with no explicit system characterization, the feedforward input required for repetitive motions of a system. The algorithm is based on the secant method of numerical analysis and gives accurate motion at frequencies limited only by the signal-to-noise ratio and the actuator power and range. We illustrate the secant-solver algorithm on a stage used for atomic force 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

Compact three-dimensional super-resolution system based on fluorescence emission difference microscopy

NASA Astrophysics Data System (ADS)

Zhu, Dazhao; Chen, Youhua; Fang, Yue; Hussain, Anwar; Kuang, Cuifang; Zhou, Xiaoxu; Xu, Yingke; Liu, Xu

2017-12-01

A compact microscope system for three-dimensional (3-D) super-resolution imaging is presented. The super-resolution capability of the system is based on a size-reduced effective 3-D point spread function generated through the fluorescence emission difference (FED) method. The appropriate polarization direction distribution and manipulation allows the panel active area of the spatial light modulator to be fully utilized. This allows simultaneous modulation of the incident light by two kinds of phase masks to be performed with a single spatial light modulator in order to generate a 3-D negative spot. The system is more compact than standard 3-D FED systems while maintaining all the advantages of 3-D FED microscopy. The experimental results demonstrated the improvement in 3-D resolution by nearly 1.7 times and 1.6 times compared to the classic confocal resolution in the lateral and axial directions, respectively.

The application of Fresnel zone plate based projection in optofluidic microscopy.

PubMed

Wu, Jigang; Cui, Xiquan; Lee, Lap Man; Yang, Changhuei

2008-09-29

Optofluidic microscopy (OFM) is a novel technique for low-cost, high-resolution on-chip microscopy imaging. In this paper we report the use of the Fresnel zone plate (FZP) based projection in OFM as a cost-effective and compact means for projecting the transmission through an OFM's aperture array onto a sensor grid. We demonstrate this approach by employing a FZP (diameter = 255 microm, focal length = 800 microm) that has been patterned onto a glass slide to project the transmission from an array of apertures (diameter = 1 microm, separation = 10 microm) onto a CMOS sensor. We are able to resolve the contributions from 44 apertures on the sensor under the illumination from a HeNe laser (wavelength = 633 nm). The imaging quality of the FZP determines the effective field-of-view (related to the number of resolvable transmissions from apertures) but not the image resolution of such an OFM system--a key distinction from conventional microscope systems. We demonstrate the capability of the integrated system by flowing the protist Euglena gracilis across the aperture array microfluidically and performing OFM imaging of the samples.

Microfluidic microscopy-assisted label-free approach for cancer screening: automated microfluidic cytology for cancer screening.

PubMed

Jagannadh, Veerendra Kalyan; Gopakumar, G; Subrahmanyam, Gorthi R K Sai; Gorthi, Sai Siva

2017-05-01

Each year, about 7-8 million deaths occur due to cancer around the world. More than half of the cancer-related deaths occur in the less-developed parts of the world. Cancer mortality rate can be reduced with early detection and subsequent treatment of the disease. In this paper, we introduce a microfluidic microscopy-based cost-effective and label-free approach for identification of cancerous cells. We outline a diagnostic framework for the same and detail an instrumentation layout. We have employed classical computer vision techniques such as 2D principal component analysis-based cell type representation followed by support vector machine-based classification. Analogous to criminal face recognition systems implemented with help of surveillance cameras, a signature-based approach for cancerous cell identification using microfluidic microscopy surveillance is demonstrated. Such a platform would facilitate affordable mass screening camps in the developing countries and therefore help decrease cancer mortality rate.

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

PubMed Central

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

2004-01-01

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

Removal of anti-Stokes emission background in STED microscopy by FPGA-based synchronous detection

NASA Astrophysics Data System (ADS)

Castello, M.; Tortarolo, G.; Coto Hernández, I.; Deguchi, T.; Diaspro, A.; Vicidomini, G.

2017-05-01

In stimulated emission depletion (STED) microscopy, the role of the STED beam is to de-excite, via stimulated emission, the fluorophores that have been previously excited by the excitation beam. This condition, together with specific beam intensity distributions, allows obtaining true sub-diffraction spatial resolution images. However, if the STED beam has a non-negligible probability to excite the fluorophores, a strong fluorescent background signal (anti-Stokes emission) reduces the effective resolution. For STED scanning microscopy, different synchronous detection methods have been proposed to remove this anti-Stokes emission background and recover the resolution. However, every method works only for a specific STED microscopy implementation. Here we present a user-friendly synchronous detection method compatible with any STED scanning microscope. It exploits a data acquisition (DAQ) card based on a field-programmable gate array (FPGA), which is progressively used in STED microscopy. In essence, the FPGA-based DAQ card synchronizes the fluorescent signal registration, the beam deflection, and the excitation beam interruption, providing a fully automatic pixel-by-pixel synchronous detection method. We validate the proposed method in both continuous wave and pulsed STED microscope systems.

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

NASA Astrophysics Data System (ADS)

Yang, Yong-fa; Li, Qi

2014-12-01

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

Numerical tilting compensation in microscopy based on wavefront sensing using transport of intensity equation method

NASA Astrophysics Data System (ADS)

Hu, Junbao; Meng, Xin; Wei, Qi; Kong, Yan; Jiang, Zhilong; Xue, Liang; Liu, Fei; Liu, Cheng; Wang, Shouyu

2018-03-01

Wide-field microscopy is commonly used for sample observations in biological research and medical diagnosis. However, the tilting error induced by the oblique location of the image recorder or the sample, as well as the inclination of the optical path often deteriorates the imaging quality. In order to eliminate the tilting in microscopy, a numerical tilting compensation technique based on wavefront sensing using transport of intensity equation method is proposed in this paper. Both the provided numerical simulations and practical experiments prove that the proposed technique not only accurately determines the tilting angle with simple setup and procedures, but also compensates the tilting error for imaging quality improvement even in the large tilting cases. Considering its simple systems and operations, as well as image quality improvement capability, it is believed the proposed method can be applied for tilting compensation in the optical microscopy.

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

NASA Astrophysics Data System (ADS)

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

2017-01-01

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

GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method

PubMed Central

Kim, Byungyeon; Park, Byungjun; Lee, Seungrag; Won, Youngjae

2016-01-01

We demonstrated GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method. Our algorithm was verified for various fluorescence lifetimes and photon numbers. The GPU processing time was faster than the physical scanning time for images up to 800 × 800, and more than 149 times faster than a single core CPU. The frame rate of our system was demonstrated to be 13 fps for a 200 × 200 pixel image when observing maize vascular tissue. This system can be utilized for observing dynamic biological reactions, medical diagnosis, and real-time industrial inspection. PMID:28018724

Simulated single molecule microscopy with SMeagol.

PubMed

Lindén, Martin; Ćurić, Vladimir; Boucharin, Alexis; Fange, David; Elf, Johan

2016-08-01

SMeagol is a software tool to simulate highly realistic microscopy data based on spatial systems biology models, in order to facilitate development, validation and optimization of advanced analysis methods for live cell single molecule microscopy data. SMeagol runs on Matlab R2014 and later, and uses compiled binaries in C for reaction-diffusion simulations. Documentation, source code and binaries for Mac OS, Windows and Ubuntu Linux can be downloaded from http://smeagol.sourceforge.net johan.elf@icm.uu.se Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

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.

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.

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

De Vos, Winnok H., E-mail: winnok.devos@uantwerpen.be; Cell Systems and Imaging Research Group, Department of Molecular Biotechnology, Ghent University, Ghent; Beghuin, Didier

As commercial space flights have become feasible and long-term extraterrestrial missions are planned, it is imperative that the impact of space travel and the space environment on human physiology be thoroughly characterized. Scrutinizing the effects of potentially detrimental factors such as ionizing radiation and microgravity at the cellular and tissue level demands adequate visualization technology. Advanced light microscopy (ALM) is the leading tool for non-destructive structural and functional investigation of static as well as dynamic biological systems. In recent years, technological developments and advances in photochemistry and genetic engineering have boosted all aspects of resolution, readout and throughput, rendering ALMmore » ideally suited for biological space research. While various microscopy-based studies have addressed cellular response to space-related environmental stressors, biological endpoints have typically been determined only after the mission, leaving an experimental gap that is prone to bias results. An on-board, real-time microscopical monitoring device can bridge this gap. Breadboards and even fully operational microscope setups have been conceived, but they need to be rendered more compact and versatile. Most importantly, they must allow addressing the impact of gravity, or the lack thereof, on physiologically relevant biological systems in space and in ground-based simulations. In order to delineate the essential functionalities for such a system, we have reviewed the pending questions in space science, the relevant biological model systems, and the state-of-the art in ALM. Based on a rigorous trade-off, in which we recognize the relevance of multi-cellular systems and the cellular microenvironment, we propose a compact, but flexible concept for space-related cell biological research that is based on light sheet microscopy.« less

Image contrast mechanisms in dynamic friction force microscopy: Antimony particles on graphite

NASA Astrophysics Data System (ADS)

Mertens, Felix; Göddenhenrich, Thomas; Dietzel, Dirk; Schirmeisen, Andre

2017-01-01

Dynamic Friction Force Microscopy (DFFM) is a technique based on Atomic Force Microscopy (AFM) where resonance oscillations of the cantilever are excited by lateral actuation of the sample. During this process, the AFM tip in contact with the sample undergoes a complex movement which consists of alternating periods of sticking and sliding. Therefore, DFFM can give access to dynamic transition effects in friction that are not accessible by alternative techniques. Using antimony nanoparticles on graphite as a model system, we analyzed how combined influences of friction and topography can effect different experimental configurations of DFFM. Based on the experimental results, for example, contrast inversion between fractional resonance and band excitation imaging strategies to extract reliable tribological information from DFFM images are devised.

Portable fiber-optic taper coupled optical microscopy platform

NASA Astrophysics Data System (ADS)

Wang, Weiming; Yu, Yan; Huang, Hui; Ou, Jinping

2017-04-01

The optical fiber taper coupled with CMOS has advantages of high sensitivity, compact structure and low distortion in the imaging platform. So it is widely used in low light, high speed and X-ray imaging systems. In the meanwhile, the peculiarity of the coupled structure can meet the needs of the demand in microscopy imaging. Toward this end, we developed a microscopic imaging platform based on the coupling of cellphone camera module and fiber optic taper for the measurement of the human blood samples and ascaris lumbricoides. The platform, weighing 70 grams, is based on the existing camera module of the smartphone and a fiber-optic array which providing a magnification factor of 6x.The top facet of the taper, on which samples are placed, serves as an irregular sampling grid for contact imaging. The magnified images of the sample, located on the bottom facet of the fiber, are then projected onto the CMOS sensor. This paper introduces the portable medical imaging system based on the optical fiber coupling with CMOS, and theoretically analyzes the feasibility of the system. The image data and process results either can be stored on the memory or transmitted to the remote medical institutions for the telemedicine. We validate the performance of this cell-phone based microscopy platform using human blood samples and test target, achieving comparable results to a standard bench-top microscope.

Analysis of multi-channel microscopy: Spectral self-interference, multi-detector confocal and 4Pi systems

NASA Astrophysics Data System (ADS)

Davis, Brynmor J.

Fluorescence microscopy is an important and ubiquitous tool in biological imaging due to the high specificity with which fluorescent molecules can be attached to an organism and the subsequent nondestructive in-vivo imaging allowed. Focused-light microscopies allow three-dimensional fluorescence imaging but their resolution is restricted by diffraction. This effect is particularly limiting in the axial dimension as the diffraction-limited focal volume produced by a lens is more extensive along the optical axis than perpendicular to it. Approaches such as confocal microscopy and 4Pi microscopy have been developed to improve the axial resolution. Spectral Self-Interference Fluorescence Microscopy (SSFM) is another high-axial-resolution technique and is the principal subject of this dissertation. Nanometer-precision localization of a single fluorescent layer has been demonstrated using SSFM. This accuracy compares favorably with the axial resolutions given by confocal and 4Pi systems at similar operating parameters (these resolutions are approximately 350nm and 80nm respectively). This theoretical work analyzes the expected performance of the SSFM system when imaging a general object, i.e. an arbitrary fluorophore density function rather than a single layer. An existing model of SSFM is used in simulations to characterize the system's resolution. Several statistically-based reconstruction methods are applied to show that the expected resolution for SSFM is similar to 4Pi microscopy for a general object but does give very high localization accuracy when the object is known to consist of a limited number of layers. SSFM is then analyzed in a linear systems framework and shown to have strong connections, both physically and mathematically, to a multi-channel 4Pi microscope. Fourier-domain analysis confirms that SSFM cannot be expected to outperform this multi-channel 4Pi instrument. Differences between the channels in spatial-scanning, multi-channel microscopies are then exploited to show that such instruments can operate at a sub-Nyquist scanning rate but still produce images largely free of aliasing effects. Multi-channel analysis is also used to show how light typically discarded in confocal and 4Pi systems can be collected and usefully incorporated into the measured image.

Multimodal fiber source for nonlinear microscopy based on a dissipative soliton laser

PubMed Central

Lamb, Erin S.; Wise, Frank W.

2015-01-01

Recent developments in high energy femtosecond fiber lasers have enabled robust and lower-cost sources for multiphoton-fluorescence and harmonic-generation imaging. However, picosecond pulses are better suited for Raman scattering microscopy, so the ideal multimodal source for nonlinear microcopy needs to provide both durations. Here we present spectral compression of a high-power femtosecond fiber laser as a route to producing transform-limited picosecond pulses. These pulses pump a fiber optical parametric oscillator to yield a robust fiber source capable of providing the synchronized picosecond pulse trains needed for Raman scattering microscopy. Thus, this system can be used as a multimodal platform for nonlinear microscopy techniques. PMID:26417497

Using nuclear microscopy to characterize the interaction of textile-used silver nanoparticles with a biological wastewater treatment system

NASA Astrophysics Data System (ADS)

Bento, J. B.; Franca, R. D. G.; Pinheiro, T.; Alves, L. C.; Pinheiro, H. M.; Lourenço, N. D.

2017-08-01

The use of engineered nanoparticles in the textile industry has been rapidly increasing but their fate during biological wastewater treatment is largely unknown. The goal of the current study was to characterize the interaction of silver nanoparticles (AgNPs), used in the textile industry, with a biological wastewater treatment system based on aerobic granular sludge (AGS). The exposure tests were performed using a laboratory-scale sequencing batch reactor (SBR) system with AGS. The behavior and fate of textile AgNPs in the AGS system was studied with nuclear microscopy techniques. Elemental maps of AGS samples collected from the SBR showed that AgNPs typically clustered in agglomerates of small dimensions (<10 μm), which were preferentially associated with extracellular polymeric substances (EPS). This preliminary study highlights the potential application of nuclear microscopy for the characterization of the behavior and fate of AgNPs in AGS. The detailed compartmentalization of AgNPs in AGS components obtained with nuclear microscopy provides new and relevant information concerning AgNPs retention. This will be important in biotechnological terms to delineate strategies for AgNPs removal from textile wastewater.

Multiphoton microscopy system with a compact fiber-based femtosecond-pulse laser and handheld probe

PubMed Central

Liu, Gangjun; Kieu, Khanh; Wise, Frank W.; Chen, Zhongping

2012-01-01

We report on the development of a compact multiphoton microscopy (MPM) system that integrates a compact and robust fiber laser with a miniature probe. The all normal dispersion fiber femtosecond laser has a central wavelength of 1.06 μm, pulse width of 125 fs and average power of more than 1 W. A double cladding photonic crystal fiber was used to deliver the excitation beam and to collect the two-photon signal. The hand-held probe included galvanometer-based mirror scanners, relay lenses and a focusing lens. The packaged probe had a diameter of 16 mm. Second harmonic generation (SHG) images and two-photon excited fluorescence (TPEF) images of biological tissues were demonstrated using the system. MPM images of different biological tissues acquired by the compact system which integrates an FBFP laser, an DCPCF and a miniature handheld probe. PMID:20635426

Development of a Fiber Laser with Independently Adjustable Properties for Optical Resolution Photoacoustic Microscopy.

PubMed

Aytac-Kipergil, Esra; Demirkiran, Aytac; Uluc, Nasire; Yavas, Seydi; Kayikcioglu, Tunc; Salman, Sarper; Karamuk, Sohret Gorkem; Ilday, Fatih Omer; Unlu, Mehmet Burcin

2016-12-08

Photoacoustic imaging is based on the detection of generated acoustic waves through thermal expansion of tissue illuminated by short laser pulses. Fiber lasers as an excitation source for photoacoustic imaging have recently been preferred for their high repetition frequencies. Here, we report a unique fiber laser developed specifically for multiwavelength photoacoustic microscopy system. The laser is custom-made for maximum flexibility in adjustment of its parameters; pulse duration (5-10 ns), pulse energy (up to 10 μJ) and repetition frequency (up to 1 MHz) independently from each other and covers a broad spectral region from 450 to 1100 nm and also can emit wavelengths of 532, 355, and 266 nm. The laser system consists of a master oscillator power amplifier, seeding two stages; supercontinuum and harmonic generation units. The laser is outstanding since the oscillator, amplifier and supercontinuum generation parts are all-fiber integrated with custom-developed electronics and software. To demonstrate the feasibility of the system, the images of several elements of standardized resolution test chart are acquired at multiple wavelengths. The lateral resolution of optical resolution photoacoustic microscopy system is determined as 2.68 μm. The developed system may pave the way for spectroscopic photoacoustic microscopy applications via widely tunable fiber laser technologies.

Development of a Fiber Laser with Independently Adjustable Properties for Optical Resolution Photoacoustic Microscopy

PubMed Central

Aytac-Kipergil, Esra; Demirkiran, Aytac; Uluc, Nasire; Yavas, Seydi; Kayikcioglu, Tunc; Salman, Sarper; Karamuk, Sohret Gorkem; Ilday, Fatih Omer; Unlu, Mehmet Burcin

2016-01-01

Photoacoustic imaging is based on the detection of generated acoustic waves through thermal expansion of tissue illuminated by short laser pulses. Fiber lasers as an excitation source for photoacoustic imaging have recently been preferred for their high repetition frequencies. Here, we report a unique fiber laser developed specifically for multiwavelength photoacoustic microscopy system. The laser is custom-made for maximum flexibility in adjustment of its parameters; pulse duration (5–10 ns), pulse energy (up to 10 μJ) and repetition frequency (up to 1 MHz) independently from each other and covers a broad spectral region from 450 to 1100 nm and also can emit wavelengths of 532, 355, and 266 nm. The laser system consists of a master oscillator power amplifier, seeding two stages; supercontinuum and harmonic generation units. The laser is outstanding since the oscillator, amplifier and supercontinuum generation parts are all-fiber integrated with custom-developed electronics and software. To demonstrate the feasibility of the system, the images of several elements of standardized resolution test chart are acquired at multiple wavelengths. The lateral resolution of optical resolution photoacoustic microscopy system is determined as 2.68 μm. The developed system may pave the way for spectroscopic photoacoustic microscopy applications via widely tunable fiber laser technologies. PMID:27929049

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

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

Cost analysis of nucleic acid amplification for diagnosing pulmonary tuberculosis, within the context of the Brazilian Unified Health Care System.

PubMed

Pinto, Márcia; Entringer, Aline Piovezan; Steffen, Ricardo; Trajman, Anete

2015-01-01

We estimated the costs of a molecular test for Mycobacterium tuberculosis and resistance to rifampin (Xpert MTB/RIF) and of smear microscopy, within the Brazilian Sistema Único de Saúde (SUS, Unified Health Care System). In SUS laboratories in the cities of Rio de Janeiro and Manaus, we performed activity-based costing and micro-costing. The mean unit costs for Xpert MTB/RIF and smear microscopy were R$35.57 and R$14.16, respectively. The major cost drivers for Xpert MTB/RIF and smear microscopy were consumables/reagents and staff, respectively. These results might facilitate future cost-effectiveness studies and inform the decision-making process regarding the expansion of Xpert MTB/RIF use in Brazil.

A simple tool for stereological assessment of digital images: the STEPanizer.

PubMed

Tschanz, S A; Burri, P H; Weibel, E R

2011-07-01

STEPanizer is an easy-to-use computer-based software tool for the stereological assessment of digitally captured images from all kinds of microscopical (LM, TEM, LSM) and macroscopical (radiology, tomography) imaging modalities. The program design focuses on providing the user a defined workflow adapted to most basic stereological tasks. The software is compact, that is user friendly without being bulky. STEPanizer comprises the creation of test systems, the appropriate display of digital images with superimposed test systems, a scaling facility, a counting module and an export function for the transfer of results to spreadsheet programs. Here we describe the major workflow of the tool illustrating the application on two examples from transmission electron microscopy and light microscopy, respectively. © 2011 The Authors Journal of Microscopy © 2011 Royal Microscopical Society.

Cost analysis of nucleic acid amplification for diagnosing pulmonary tuberculosis, within the context of the Brazilian Unified Health Care System

PubMed Central

Pinto, Márcia; Entringer, Aline Piovezan; Steffen, Ricardo; Trajman, Anete

2015-01-01

ABSTRACT We estimated the costs of a molecular test for Mycobacterium tuberculosis and resistance to rifampin (Xpert MTB/RIF) and of smear microscopy, within the Brazilian Sistema Único de Saúde (SUS, Unified Health Care System). In SUS laboratories in the cities of Rio de Janeiro and Manaus, we performed activity-based costing and micro-costing. The mean unit costs for Xpert MTB/RIF and smear microscopy were R$35.57 and R$14.16, respectively. The major cost drivers for Xpert MTB/RIF and smear microscopy were consumables/reagents and staff, respectively. These results might facilitate future cost-effectiveness studies and inform the decision-making process regarding the expansion of Xpert MTB/RIF use in Brazil. PMID:26785963

Cost-effectiveness of diagnostic for malaria in Extra-Amazon Region, Brazil

PubMed Central

2012-01-01

Background Rapid diagnostic tests (RDT) for malaria have been demonstrated to be effective and they should replace microscopy in certain areas. Method The cost-effectiveness of five RDT and thick smear microscopy was estimated and compared. Data were collected on Brazilian Extra-Amazon Region. Data sources included the National Malaria Control Programme of the Ministry of Health, the National Healthcare System reimbursement table, laboratory suppliers and scientific literature. The perspective was that of the Brazilian public health system, the analytical horizon was from the start of fever until the diagnostic results provided to patient and the temporal reference was that of year 2010. Two costing methods were produced, based on exclusive-use microscopy or shared-use microscopy. The results were expressed in costs per adequately diagnosed cases in 2010 U.S. dollars. One-way sensitivity analysis was performed considering key model parameters. Results In the cost-effectiveness analysis with exclusive-use microscopy, the RDT CareStart™ was the most cost-effective diagnostic strategy. Microscopy was the most expensive and most effective, with an additional case adequately diagnosed by microscopy costing US$ 35,550.00 in relation to CareStart™. In opposite, in the cost-effectiveness analysis with shared-use microscopy, the thick smear was extremely cost-effective. Introducing into the analytic model with shared-use microscopy a probability for individual access to the diagnosis, assuming a probability of 100% of access for a public health system user to any RDT and, hypothetically, of 85% of access to microscopy, this test saw its effectiveness reduced and was dominated by the RDT CareStart™. Conclusion The analysis of cost-effectiveness of malaria diagnosis technologies in the Brazilian Extra-Amazon Region depends on the exclusive or shared use of the microscopy. Following the assumptions of this study, shared-use microscopy would be the most cost-effective strategy of the six technologies evaluated. However, if used exclusively for diagnosing malaria, microscopy would be the worst use of resources. Microscopy would not be the most cost-effective strategy, even when structure is shared with other programmes, when the probability of a patient having access to it was reduced. Under these circumstances, the RDT CareStart™ would be the most cost-effective strategy. PMID:23176717

CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography).

PubMed

Siegel, Nisan; Storrie, Brian; Bruce, Marc; Brooker, Gary

2015-02-07

FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called "CINCH". An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution.

Image correlation microscopy for uniform illumination.

PubMed

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

2010-01-01

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

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.

Quartz tuning fork-based frequency modulation atomic force spectroscopy and microscopy with all digital phase-locked loop

NASA Astrophysics Data System (ADS)

An, Sangmin; Hong, Mun-heon; Kim, Jongwoo; Kwon, Soyoung; Lee, Kunyoung; Lee, Manhee; Jhe, Wonho

2012-11-01

We present a platform for the quartz tuning fork (QTF)-based, frequency modulation atomic force microscopy (FM-AFM) system for quantitative study of the mechanical or topographical properties of nanoscale materials, such as the nano-sized water bridge formed between the quartz tip (˜100 nm curvature) and the mica substrate. A thermally stable, all digital phase-locked loop is used to detect the small frequency shift of the QTF signal resulting from the nanomaterial-mediated interactions. The proposed and demonstrated novel FM-AFM technique provides high experimental sensitivity in the measurement of the viscoelastic forces associated with the confined nano-water meniscus, short response time, and insensitivity to amplitude noise, which are essential for precision dynamic force spectroscopy and microscopy.

Quartz tuning fork-based frequency modulation atomic force spectroscopy and microscopy with all digital phase-locked loop.

PubMed

An, Sangmin; Hong, Mun-heon; Kim, Jongwoo; Kwon, Soyoung; Lee, Kunyoung; Lee, Manhee; Jhe, Wonho

2012-11-01

We present a platform for the quartz tuning fork (QTF)-based, frequency modulation atomic force microscopy (FM-AFM) system for quantitative study of the mechanical or topographical properties of nanoscale materials, such as the nano-sized water bridge formed between the quartz tip (~100 nm curvature) and the mica substrate. A thermally stable, all digital phase-locked loop is used to detect the small frequency shift of the QTF signal resulting from the nanomaterial-mediated interactions. The proposed and demonstrated novel FM-AFM technique provides high experimental sensitivity in the measurement of the viscoelastic forces associated with the confined nano-water meniscus, short response time, and insensitivity to amplitude noise, which are essential for precision dynamic force spectroscopy and microscopy.

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

PubMed

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

2010-12-20

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

Observation of the immune response of cells and tissue through multimodal label-free microscopy

NASA Astrophysics Data System (ADS)

Pavillon, Nicolas; Smith, Nicholas I.

2017-02-01

We present applications of a label-free approach to assess the immune response based on the combination of interferometric microscopy and Raman spectroscopy, which makes it possible to simultaneously acquire morphological and molecular information of live cells. We employ this approach to derive statistical models for predicting the activation state of macrophage cells based both on morphological parameters extracted from the high-throughput full-field quantitative phase imaging, and on the molecular content information acquired through Raman spectroscopy. We also employ a system for 3D imaging based on coherence gating, enabling specific targeting of the Raman channel to structures of interest within tissue.

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.

An electromechanical material testing system for in situ electron microscopy and applications.

PubMed

Zhu, Yong; Espinosa, Horacio D

2005-10-11

We report the development of a material testing system for in situ electron microscopy (EM) mechanical testing of nanostructures. The testing system consists of an actuator and a load sensor fabricated by means of surface micromachining. This previously undescribed nanoscale material testing system makes possible continuous observation of the specimen deformation and failure with subnanometer resolution, while simultaneously measuring the applied load electronically with nanonewton resolution. This achievement was made possible by the integration of electromechanical and thermomechanical components based on microelectromechanical system technology. The system capabilities are demonstrated by the in situ EM testing of free-standing polysilicon films, metallic nanowires, and carbon nanotubes. In particular, a previously undescribed real-time instrumented in situ transmission EM observation of carbon nanotubes failure under tensile load is presented here.

Synchrotron-based X-ray microscopic studies for bioeffects of nanomaterials.

PubMed

Zhu, Ying; Cai, Xiaoqing; Li, Jiang; Zhong, Zengtao; Huang, Qing; Fan, Chunhai

2014-04-01

There have been increasing interests in studying biological effects of nanomaterials, which are nevertheless faced up with many challenges due to the nanoscale dimensions and unique chemical properties of nanomaterials. Synchrotron-based X-ray microscopy, an advanced imaging technology with high spatial resolution and excellent elemental specificity, provides a new platform for studying interactions between nanomaterials and living systems. In this article, we review the recent progress of X-ray microscopic studies on bioeffects of nanomaterials in several living systems including cells, model organisms, animals and plants. We aim to provide an overview of the state of the art, and the advantages of using synchrotron-based X-ray microscopy for characterizing in vitro and in vivo behaviors and biodistribution of nanomaterials. We also expect that the use of a combination of new synchrotron techniques should offer unprecedented opportunities for better understanding complex interactions at the nano-biological interface and accounting for unique bioeffects of nanomaterials. Synchrotron-based X-ray microscopy is a non-destructive imaging technique that enables high resolution spatial mapping of metals with elemental level detection methods. This review summarizes the current use and perspectives of this novel technique in studying the biology and tissue interactions of nanomaterials. Copyright © 2014 Elsevier Inc. All rights reserved.

Cost-Effectiveness of Automated Digital Microscopy for Diagnosis of Active Tuberculosis.

PubMed

Jha, Swati; Ismail, Nazir; Clark, David; Lewis, James J; Omar, Shaheed; Dreyer, Andries; Chihota, Violet; Churchyard, Gavin; Dowdy, David W

2016-01-01

Automated digital microscopy has the potential to improve the diagnosis of tuberculosis (TB), particularly in settings where molecular testing is too expensive to perform routinely. The cost-effectiveness of TB diagnostic algorithms using automated digital microscopy remains uncertain. Using data from a demonstration study of an automated digital microscopy system (TBDx, Applied Visual Systems, Inc.), we performed an economic evaluation of TB diagnosis in South Africa from the health system perspective. The primary outcome was the incremental cost per new TB diagnosis made. We considered costs and effectiveness of different algorithms for automated digital microscopy, including as a stand-alone test and with confirmation of positive results with Xpert MTB/RIF ('Xpert', Cepheid, Inc.). Results were compared against both manual microscopy and universal Xpert testing. In settings willing to pay $2000 per incremental TB diagnosis, universal Xpert was the preferred strategy. However, where resources were not sufficient to support universal Xpert, and a testing volume of at least 30 specimens per day could be ensured, automated digital microscopy with Xpert confirmation of low-positive results could facilitate the diagnosis of 79-84% of all Xpert-positive TB cases, at 50-60% of the total cost. The cost-effectiveness of this strategy was $1280 per incremental TB diagnosis (95% uncertainty range, UR: $340-$3440) in the base case, but improved under conditions likely reflective of many settings in sub-Saharan Africa: $677 per diagnosis (95% UR: $450-$935) when sensitivity of manual smear microscopy was lowered to 0.5, and $956 per diagnosis (95% UR: $40-$2910) when the prevalence of multidrug-resistant TB was lowered to 1%. Although universal Xpert testing is the preferred algorithm for TB diagnosis when resources are sufficient, automated digital microscopy can identify the majority of cases and halve the cost of diagnosis and treatment when resources are more scarce and multidrug-resistant TB is not common.

Fluorescence confocal microscopy for pathologists.

PubMed

Ragazzi, Moira; Piana, Simonetta; Longo, Caterina; Castagnetti, Fabio; Foroni, Monica; Ferrari, Guglielmo; Gardini, Giorgio; Pellacani, Giovanni

2014-03-01

Confocal microscopy is a non-invasive method of optical imaging that may provide microscopic images of untreated tissue that correspond almost perfectly to hematoxylin- and eosin-stained slides. Nowadays, following two confocal imaging systems are available: (1) reflectance confocal microscopy, based on the natural differences in refractive indices of subcellular structures within the tissues; (2) fluorescence confocal microscopy, based on the use of fluorochromes, such as acridine orange, to increase the contrast epithelium-stroma. In clinical practice to date, confocal microscopy has been used with the goal of obviating the need for excision biopsies, thereby reducing the need for pathological examination. The aim of our study was to test fluorescence confocal microscopy on different types of surgical specimens, specifically breast, lymph node, thyroid, and colon. The confocal images were correlated to the corresponding histological sections in order to provide a morphologic parallel and to highlight current limitations and possible applications of this technology for surgical pathology practice. As a result, neoplastic tissues were easily distinguishable from normal structures and reactive processes such as fibrosis; the use of fluorescence enhanced contrast and image quality in confocal microscopy without compromising final histologic evaluation. Finally, the fluorescence confocal microscopy images of the adipose tissue were as accurate as those of conventional histology and were devoid of the frozen-section-related artefacts that can compromise intraoperative evaluation. Despite some limitations mainly related to black/white images, which require training in imaging interpretation, this study confirms that fluorescence confocal microscopy may represent an alternative to frozen sections in the assessment of margin status in selected settings or when the conservation of the specimen is crucial. This is the first study to employ fluorescent confocal microscopy on surgical specimens other than the skin and to evaluate the diagnostic capability of this technology from pathologists' viewpoint.

Electronically tunable femtosecond all-fiber optical parametric oscillator for multi-photon microscopy

NASA Astrophysics Data System (ADS)

Hellwig, Tim; Brinkmann, Maximilian; Fallnich, Carsten

2018-02-01

We present a femtosecond fiber-based optical parametric oscillator (FOPO) for multiphoton microscopy with wavelength tuning by electronic repetition rate tuning in combination with a dispersive filter in the FOPO cavity. The all-spliced, all-fiber FOPO cavity is based on polarization-maintaining fibers and a broadband output coupler, allowing to get access to the resonant signal pulses as well as the idler pulses simultaneously. The system was pumped by a gain-switched fiber-coupled laser diode emitting pulses at a central wavelength of 1030 nm and an electronically tunable repetition frequency of about 2 MHz. The pump pulses were amplified in an Ytterbium fiber amplifier system with a pulse duration after amplification of 13 ps. Tuning of the idler (1140 nm - 1300 nm) and signal wavelengths (850 nm - 940 nm) was achieved by changing the repetition frequency of the pump laser by about 4 kHz. The generated signal pulses reached a pulse energy of up to 9.2 nJ at 920 nm and were spectrally broadened to about 6 nm in the FOPO by a combination of self-phase and cross-phase modulation. We showed external compression of the idler pulses at 920 nm to about 430 fs and appleid them to two-photon excitation microscopy with green fluorescent dyes. The presented system constitutes an important step towards a fully fiber-integrated all-electronically tunable and, thereby, programmable light source and already embodies a versatile and flexible light source for applications, e.g., for smart microscopy.

All fiber nonlinear microscopy at 1550 nm using a double-clad fiber coupler

NASA Astrophysics Data System (ADS)

Perrillat-Bottonet, Thomas; Strupler, Mathias; Leduc, Mikael; Majeau, Lucas; Godbout, Nicolas; Boudoux, Caroline

2017-02-01

Nonlinear microscopy has already shown its impact in biological research, namely in the fields of neurobiology, immunology, cancer research and embryology. Typically, these microscopes operate under free space propagation, using a dichroic mirror to separate the nonlinear signals from the excitation laser. While powerful such implementations are difficult to translate from the laboratory to a clinical setting where the environment is less controlled. Therefore, we propose an alignment-free all-fiber nonlinear microscopy system at 1550 nm based on double-clad fibers (DCF). As sectioning is performed through nonlinear effects, nonlinear microscopy does not require a detection pinhole, and. the DCF inner cladding can be used for efficient collection of nonlinear signals. The built system allows for multiplexing second harmonic generation (SHG) and two-photon excitation fluorescence (2PEF), collected from the inner cladding; and reflectance confocal microscopy (RCM), detected from the core acting as the confocal pinhole. Finally, an asymmetric double-clad fiber coupler (DCFC) is used to address efficiently both DCF channels. This all-fiber system is more compact and less sensitive to alignment, but requires carefully managing the transmission of the femtosecond pulse in the fiber. This is addressed using dispersion compensation fiber, pulse compression and solitonic propagation. Additionally, with a source centered at 1550 nm, we benefit from reduced sample scattering thus increasing the depth of field in comparison with systems operating at 800 nm. Overall we believe that the developed system could be transferred in clinics to enable in-vivo and in-situ imaging of human patient.

PSD microscopy: a new technique for adaptive local scanning of microscale objects.

PubMed

Rahimi, Mehdi; Shen, Yantao

2017-01-01

A position-sensitive detector/device (PSD) is a sensor that is capable of tracking the location of a laser beam on its surface. PSDs are used in many scientific instruments and technical applications including but not limited to atomic force microscopy, human eye movement monitoring, mirrors or machine tool alignment, vibration analysis, beam position control and so on. This work intends to propose a new application using the PSD. That is a new microscopy system called scanning PSD microscopy. The working mechanism is about putting an object on the surface of the PSD and fast scanning its area with a laser beam. To achieve a high degree of accuracy and precision, a reliable framework was designed using the PSD. In this work, we first tried to improve the PSD reading and its measurement performance. This was done by minimizing the effects of noise, distortion and other disturbing parameters. After achieving a high degree of confidence, the microscopy system can be implemented based on the improved PSD measurement performance. Later to improve the scanning efficiency, we developed an adaptive local scanning system to scan the whole area of the PSD in a short matter of time. It was validated that our comprehensive and adaptive local scanning method can shorten the scanning time in order of hundreds of times in comparison with the traditional raster scanning without losing any important information about the scanned 2D objects. Methods are also introduced to scan very complicated objects with bifurcations and crossings. By incorporating all these methods, the new microscopy system is capable of scanning very complicated objects in the matter of a few seconds with a resolution that is in order of a few micrometers.

Correlative microscopy of detergent granules.

PubMed

van Dalen, G; Nootenboom, P; Heussen, P C M

2011-03-01

The microstructure of detergent products for textile cleaning determines to a large extent the physical properties of these products. Correlative microscopy was used to reveal the microstructure by reconciling images obtained by scanning electron microscopy with energy dispersive X-ray analysis, X-ray microtomography and Fourier transform infrared microscopy. These techniques were applied on the same location of a subsample of a spray-dried detergent base powder embedded in polyacrylate. In this way, the three-dimensional internal and external structure of detergent granules could be investigated from milli to nano scale with detailed spatial information about the components present. This will generate knowledge how to design optimal microstructures for laundry products to obtain product properties demanded by the market. This method is also very useful for other powder systems used in a large variety of industries (e.g. for pharmaceutical, food, ceramic and metal industries). © 2010 The Authors Journal of Microscopy © 2010 The Royal Microscopical Society.

Automated Analysis of Fluorescence Microscopy Images to Identify Protein-Protein Interactions

DOE PAGES

Venkatraman, S.; Doktycz, M. J.; Qi, H.; ...

2006-01-01

The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction.more » Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors.« less

Camera array based light field microscopy

PubMed Central

Lin, Xing; Wu, Jiamin; Zheng, Guoan; Dai, Qionghai

2015-01-01

This paper proposes a novel approach for high-resolution light field microscopy imaging by using a camera array. In this approach, we apply a two-stage relay system for expanding the aperture plane of the microscope into the size of an imaging lens array, and utilize a sensor array for acquiring different sub-apertures images formed by corresponding imaging lenses. By combining the rectified and synchronized images from 5 × 5 viewpoints with our prototype system, we successfully recovered color light field videos for various fast-moving microscopic specimens with a spatial resolution of 0.79 megapixels at 30 frames per second, corresponding to an unprecedented data throughput of 562.5 MB/s for light field microscopy. We also demonstrated the use of the reported platform for different applications, including post-capture refocusing, phase reconstruction, 3D imaging, and optical metrology. PMID:26417490

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

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

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

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

PubMed

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

2015-01-01

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

Optimal model-based sensorless adaptive optics for epifluorescence microscopy.

PubMed

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

2018-01-01

We report on a universal sample-independent sensorless adaptive optics method, based on modal optimization of the second moment of the fluorescence emission from a point-like excitation. Our method employs a sample-independent precalibration, performed only once for the particular system, to establish the direct relation between the image quality and the aberration. The method is potentially applicable to any form of microscopy with epifluorescence detection, including the practically important case of incoherent fluorescence emission from a three dimensional object, through minor hardware modifications. We have applied the technique successfully to a widefield epifluorescence microscope and to a multiaperture confocal microscope.

Modeling the electrostatic field localization in nanostructures based on DLC films using the tunneling microscopy methods

NASA Astrophysics Data System (ADS)

Yakunin, Alexander N.; Aban'shin, Nikolay P.; Avetisyan, Yuri A.; Akchurin, Georgy G.; Akchurin, Garif G.

2018-04-01

A model for calculating the electrostatic field in the system "probe of a tunnel microscope - a nanostructure based on a DLC film" was developed. A finite-element modeling of the localization of the field was carried out, taking into account the morphological and topological features of the nanostructure. The obtained results and their interpretation contribute to the development of the concepts to the model of tunnel electric transport processes. The possibility for effective usage of the tunneling microscopy methods in the development of new nanophotonic devices is shown.

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

PubMed Central

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

2010-01-01

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

The quest for four-dimensional imaging in plant cell biology: it's just a matter of time

PubMed Central

Domozych, David S.

2012-01-01

Background Analysis of plant cell dynamics over time, or four-dimensional imaging (4-DI), represents a major goal of plant science. The ability to resolve structures in the third dimension within the cell or tissue during developmental events or in response to environmental or experimental stresses (i.e. 4-DI) is critical to our understanding of gene expression, post-expression modulations of macromolecules and sub-cellular system interactions. Scope Microscopy-based technologies have been profoundly integral to this type of investigation, and new and refined microscopy technologies now allow for the visualization of cell dynamics with unprecedented resolution, contrast and experimental versatility. However, certain realities of light and electron microscopy, choice of specimen and specimen preparation techniques limit the scope of readily attaining 4-DI. Today, the plant microscopist must use a combinatorial strategy whereby multiple microscopy-based investigations are used. Modern fluorescence, confocal laser scanning, transmission electron and scanning electron microscopy provide effective conduits for synthesizing data detailing live cell dynamics and highly resolved snapshots of specific cell structures that will ultimately lead to 4-DI. This review provides a synopsis of such technologies available. PMID:22628381

CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography)

PubMed Central

Siegel, Nisan; Storrie, Brian; Bruce, Marc

2016-01-01

FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called “CINCH”. An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution. PMID:26839443

A targeted drug delivery system based on dopamine functionalized nano graphene oxide

NASA Astrophysics Data System (ADS)

Masoudipour, Elham; Kashanian, Soheila; Maleki, Nasim

2017-01-01

The cellular targeting property of a biocompatible drug delivery system can widely increase the therapeutic effect against various diseases. Here, we report a dopamine conjugated nano graphene oxide (DA-nGO) carrier for cellular delivery of the anticancer drug, Methotrexate (MTX) into DA receptor positive human breast adenocarcinoma cell line. The material was characterized using scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy and UV-vis spectroscopy. Furthermore, the antineoplastic action of MTX loaded DA-nGO against DA receptor positive and negative cell lines were explored. The results presented in this article demonstrated that the application of DA functionalized GO as a targeting drug carrier can improve the drug delivery efficacy for DA receptor positive cancer cell lines and promise future designing of carrier conjugates based on it.

Development of a DNA Sensor Based on Nanoporous Pt-Rich Electrodes

NASA Astrophysics Data System (ADS)

Van Hao, Pham; Thanh, Pham Duc; Xuan, Chu Thi; Hai, Nguyen Hoang; Tuan, Mai Anh

2017-06-01

Nanoporous Pt-rich electrodes with 72 at.% Pt composition were fabricated by sputtering a Pt-Ag alloy, followed by an electrochemical dealloying process to selectively etch away Ag atoms. The surface properties of nanoporous membranes were investigated by energy-dispersive x-ray spectroscopy (EDS), scanning electron microscopy (SEM), atomic force microscopy (AFM), a documentation system, and a gel image system (Gel Doc Imager). A single strand of probe deoxyribonucleic acid (DNA) was immobilized onto the electrode surface by physical adsorption. The DNA probe and target hybridization were measured using a lock-in amplifier and an electrochemical impedance spectroscope (EIS). The nanoporous Pt-rich electrode-based DNA sensor offers a fast response time of 3.7 s, with a limit of detection (LOD) of 4.35 × 10-10 M of DNA target.

Development of targeted STORM for super resolution imaging of biological samples using digital micro-mirror device

NASA Astrophysics Data System (ADS)

Valiya Peedikakkal, Liyana; Steventon, Victoria; Furley, Andrew; Cadby, Ashley J.

2017-12-01

We demonstrate a simple illumination system based on a digital mirror device which allows for fine control over the power and pattern of illumination. We apply this to localization microscopy (LM), specifically stochastic optical reconstruction microscopy (STORM). Using this targeted STORM, we were able to image a selected area of a labelled cell without causing photo-damage to the surrounding areas of the cell.

Cost effectiveness of OptiMal® rapid diagnostic test for malaria in remote areas of the Amazon Region, Brazil

PubMed Central

2010-01-01

Background In areas with limited structure in place for microscopy diagnosis, rapid diagnostic tests (RDT) have been demonstrated to be effective. Method The cost-effectiveness of the Optimal® and thick smear microscopy was estimated and compared. Data were collected on remote areas of 12 municipalities in the Brazilian Amazon. Data sources included the National Malaria Control Programme of the Ministry of Health, the National Healthcare System reimbursement table, hospitalization records, primary data collected from the municipalities, and scientific literature. The perspective was that of the Brazilian public health system, the analytical horizon was from the start of fever until the diagnostic results provided to patient and the temporal reference was that of year 2006. The results were expressed in costs per adequately diagnosed cases in 2006 U.S. dollars. Sensitivity analysis was performed considering key model parameters. Results In the case base scenario, considering 92% and 95% sensitivity for thick smear microscopy to Plasmodium falciparum and Plasmodium vivax, respectively, and 100% specificity for both species, thick smear microscopy is more costly and more effective, with an incremental cost estimated at US$549.9 per adequately diagnosed case. In sensitivity analysis, when sensitivity and specificity of microscopy for P. vivax were 0.90 and 0.98, respectively, and when its sensitivity for P. falciparum was 0.83, the RDT was more cost-effective than microscopy. Conclusion Microscopy is more cost-effective than OptiMal® in these remote areas if high accuracy of microscopy is maintained in the field. Decision regarding use of rapid tests for diagnosis of malaria in these areas depends on current microscopy accuracy in the field. PMID:20937094

Automated quantitative cytological analysis using portable microfluidic microscopy.

PubMed

Jagannadh, Veerendra Kalyan; Murthy, Rashmi Sreeramachandra; Srinivasan, Rajesh; Gorthi, Sai Siva

2016-06-01

In this article, a portable microfluidic microscopy based approach for automated cytological investigations is presented. Inexpensive optical and electronic components have been used to construct a simple microfluidic microscopy system. In contrast to the conventional slide-based methods, the presented method employs microfluidics to enable automated sample handling and image acquisition. The approach involves the use of simple in-suspension staining and automated image acquisition to enable quantitative cytological analysis of samples. The applicability of the presented approach to research in cellular biology is shown by performing an automated cell viability assessment on a given population of yeast cells. Further, the relevance of the presented approach to clinical diagnosis and prognosis has been demonstrated by performing detection and differential assessment of malaria infection in a given sample. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pulse splitter-based nonlinear microscopy for live-cardiomyocyte imaging

PubMed Central

Wang, Zhonghai; Qin, Wan; Shao, Yonghong; Ma, Siyu; Borg, Thomas K.; Gao, Bruce Z.

2015-01-01

Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na Ji et al. in our two photon excitation fluorescence (TPEF) and SHG hybrid microscope. The system dramatically reduced photodamage to neonatal cardiomyocytes in early stages of culture, greatly increasing cell viability. Thus continuous imaging of live cardiomyocytes was achieved with a stronger laser and for a longer period than has been reported in the literature. The pulse splitter-based TPEF-SHG microscope constructed in this study was demonstrated to be an ideal imaging system for sarcomeric addition-related investigations of neonatal cardiomyocytes in early stages of culture. PMID:25767692

Atomic-resolution single-spin magnetic resonance detection concept based on tunneling force microscopy

NASA Astrophysics Data System (ADS)

Payne, A.; Ambal, K.; Boehme, C.; Williams, C. C.

2015-05-01

A study of a force detected single-spin magnetic resonance measurement concept with atomic spatial resolution is presented. The method is based upon electrostatic force detection of spin-selection rule controlled single-electron tunneling between two electrically isolated paramagnetic states. Single-spin magnetic resonance detection is possible by measuring the force detected tunneling charge noise on and off spin resonance. Simulation results of this charge noise, based upon physical models of the tunneling and spin physics, are directly compared to measured atomic force microscopy system noise. The results show that the approach could provide single-spin measurement of electrically isolated qubit states with atomic spatial resolution at room temperature.

Color calibration and fusion of lens-free and mobile-phone microscopy images for high-resolution and accurate color reproduction.

PubMed

Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

2016-06-10

Lens-free holographic microscopy can achieve wide-field imaging in a cost-effective and field-portable setup, making it a promising technique for point-of-care and telepathology applications. However, due to relatively narrow-band sources used in holographic microscopy, conventional colorization methods that use images reconstructed at discrete wavelengths, corresponding to e.g., red (R), green (G) and blue (B) channels, are subject to color artifacts. Furthermore, these existing RGB colorization methods do not match the chromatic perception of human vision. Here we present a high-color-fidelity and high-resolution imaging method, termed "digital color fusion microscopy" (DCFM), which fuses a holographic image acquired at a single wavelength with a color-calibrated image taken by a low-magnification lens-based microscope using a wavelet transform-based colorization method. We demonstrate accurate color reproduction of DCFM by imaging stained tissue sections. In particular we show that a lens-free holographic microscope in combination with a cost-effective mobile-phone-based microscope can generate color images of specimens, performing very close to a high numerical-aperture (NA) benchtop microscope that is corrected for color distortions and chromatic aberrations, also matching the chromatic response of human vision. This method can be useful for wide-field imaging needs in telepathology applications and in resource-limited settings, where whole-slide scanning microscopy systems are not available.

Photoacoustic microscopy imaging for microneedle drug delivery

NASA Astrophysics Data System (ADS)

Moothanchery, Mohesh; Seeni, Razina Z.; Xu, Chenjie; Pramanik, Manojit

2018-02-01

The recent development of novel transdermal drug delivery systems (TDDS) using microneedle technology allows micron-sized conduits to be formed within the outermost skin layers attracting keen interest in skin as an interface for localized and systemic delivery of therapeutics. In light of this, researchers are using microneedles as tools to deliver nanoparticle formulations to targeted sites for effective therapy. However, in such studies the use of traditional histological methods are employed for characterization and do not allow for the in vivo visualization of drug delivery mechanism. Hence, this study presents a novel imaging technology to characterize microneedle based nanoparticle delivery systems using optical resolution-photoacoustic microscopy (OR-PAM). In this study in vivo transdermal delivery of gold nanoparticles using microneedles in mice ear and the spatial distribution of the nanoparticles in the tissue was successfully illustrated. Characterization of parameters that are relevant in drug delivery studies such as penetration depth, efficiency of delivered gold nanoparticles were monitored using the system. Photoacoustic microscopy proves an ideal tool for the characterization studies of microneedle properties and the studies shows microneedles as an ideal tool for precise and controlled drug delivery.

Crystal plasticity finite element analysis of deformation behaviour in SAC305 solder joint

NASA Astrophysics Data System (ADS)

Darbandi, Payam

Due to the awareness of the potential health hazards associated with the toxicity of lead (Pb), actions have been taken to eliminate or reduce the use of Pb in consumer products. Among those, tin (Sn) solders have been used for the assembly of electronic systems. Anisotropy is of significant importance in all structural metals, but this characteristic is unusually strong in Sn, making Sn based solder joints one of the best examples of the influence of anisotropy. The effect of anisotropy arising from the crystal structure of tin and large grain microstructure on the microstructure and the evolution of constitutive responses of microscale SAC305 solder joints is investigated. Insights into the effects of key microstructural features and dominant plastic deformation mechanisms influencing the measured relative activity of slip systems in SAC305 are obtained from a combination of optical microscopy, orientation imaging microscopy (OIM), slip plane trace analysis and crystal plasticity finite element (CPFE) modeling. Package level SAC305 specimens were subjected to shear deformation in sequential steps and characterized using optical microscopy and OIM to identify the activity of slip systems. X-ray micro Laue diffraction and high energy monochromatic X-ray beam were employed to characterize the joint scale tensile samples to provide necessary information to be able to compare and validate the CPFE model. A CPFE model was developed that can account for relative ease of activating slip systems in SAC305 solder based upon the statistical estimation based on correlation between the critical resolved shear stress and the probability of activating various slip systems. The results from simulations show that the CPFE model developed using the statistical analysis of activity of slip system not only can satisfy the requirements associated with kinematic of plastic deformation in crystal coordinate systems (activity of slip systems) and global coordinate system (shape changes) but also this model is able to predict the evolution of stress in joint level SAC305 sample.

Towards a precise test for malaria diagnosis in the Brazilian Amazon: comparison among field microscopy, a rapid diagnostic test, nested PCR, and a computational expert system based on artificial neural networks

PubMed Central

2010-01-01

Background Accurate malaria diagnosis is mandatory for the treatment and management of severe cases. Moreover, individuals with asymptomatic malaria are not usually screened by health care facilities, which further complicates disease control efforts. The present study compared the performances of a malaria rapid diagnosis test (RDT), the thick blood smear method and nested PCR for the diagnosis of symptomatic malaria in the Brazilian Amazon. In addition, an innovative computational approach was tested for the diagnosis of asymptomatic malaria. Methods The study was divided in two parts. For the first part, passive case detection was performed in 311 individuals with malaria-related symptoms from a recently urbanized community in the Brazilian Amazon. A cross-sectional investigation compared the diagnostic performance of the RDT Optimal-IT, nested PCR and light microscopy. The second part of the study involved active case detection of asymptomatic malaria in 380 individuals from riverine communities in Rondônia, Brazil. The performances of microscopy, nested PCR and an expert computational system based on artificial neural networks (MalDANN) using epidemiological data were compared. Results Nested PCR was shown to be the gold standard for diagnosis of both symptomatic and asymptomatic malaria because it detected the major number of cases and presented the maximum specificity. Surprisingly, the RDT was superior to microscopy in the diagnosis of cases with low parasitaemia. Nevertheless, RDT could not discriminate the Plasmodium species in 12 cases of mixed infections (Plasmodium vivax + Plasmodium falciparum). Moreover, the microscopy presented low performance in the detection of asymptomatic cases (61.25% of correct diagnoses). The MalDANN system using epidemiological data was worse that the light microscopy (56% of correct diagnoses). However, when information regarding plasma levels of interleukin-10 and interferon-gamma were inputted, the MalDANN performance sensibly increased (80% correct diagnoses). Conclusions An RDT for malaria diagnosis may find a promising use in the Brazilian Amazon integrating a rational diagnostic approach. Despite the low performance of the MalDANN test using solely epidemiological data, an approach based on neural networks may be feasible in cases where simpler methods for discriminating individuals below and above threshold cytokine levels are available. PMID:20459613

Towards a precise test for malaria diagnosis in the Brazilian Amazon: comparison among field microscopy, a rapid diagnostic test, nested PCR, and a computational expert system based on artificial neural networks.

PubMed

Andrade, Bruno B; Reis-Filho, Antonio; Barros, Austeclino M; Souza-Neto, Sebastião M; Nogueira, Lucas L; Fukutani, Kiyoshi F; Camargo, Erney P; Camargo, Luís M A; Barral, Aldina; Duarte, Angelo; Barral-Netto, Manoel

2010-05-06

Accurate malaria diagnosis is mandatory for the treatment and management of severe cases. Moreover, individuals with asymptomatic malaria are not usually screened by health care facilities, which further complicates disease control efforts. The present study compared the performances of a malaria rapid diagnosis test (RDT), the thick blood smear method and nested PCR for the diagnosis of symptomatic malaria in the Brazilian Amazon. In addition, an innovative computational approach was tested for the diagnosis of asymptomatic malaria. The study was divided in two parts. For the first part, passive case detection was performed in 311 individuals with malaria-related symptoms from a recently urbanized community in the Brazilian Amazon. A cross-sectional investigation compared the diagnostic performance of the RDT Optimal-IT, nested PCR and light microscopy. The second part of the study involved active case detection of asymptomatic malaria in 380 individuals from riverine communities in Rondônia, Brazil. The performances of microscopy, nested PCR and an expert computational system based on artificial neural networks (MalDANN) using epidemiological data were compared. Nested PCR was shown to be the gold standard for diagnosis of both symptomatic and asymptomatic malaria because it detected the major number of cases and presented the maximum specificity. Surprisingly, the RDT was superior to microscopy in the diagnosis of cases with low parasitaemia. Nevertheless, RDT could not discriminate the Plasmodium species in 12 cases of mixed infections (Plasmodium vivax + Plasmodium falciparum). Moreover, the microscopy presented low performance in the detection of asymptomatic cases (61.25% of correct diagnoses). The MalDANN system using epidemiological data was worse that the light microscopy (56% of correct diagnoses). However, when information regarding plasma levels of interleukin-10 and interferon-gamma were inputted, the MalDANN performance sensibly increased (80% correct diagnoses). An RDT for malaria diagnosis may find a promising use in the Brazilian Amazon integrating a rational diagnostic approach. Despite the low performance of the MalDANN test using solely epidemiological data, an approach based on neural networks may be feasible in cases where simpler methods for discriminating individuals below and above threshold cytokine levels are available.

Comparison of the Automated cobas u 701 Urine Microscopy and UF-1000i Flow Cytometry Systems and Manual Microscopy in the Examination of Urine Sediments.

PubMed

Lee, Wonmok; Ha, Jung-Sook; Ryoo, Nam-Hee

2016-09-01

The cobas u 701, a new automated image-based urine sediment analyzer, was introduced recently. In this study, we compared its performance with that of UF-1000i flow cytometry and manual microscopy in the examination of urine sediments. Precision, linearity, and carry-over were determined for the two urine sediment analyzers. For a comparison of the method, 300 urine samples were examined by the automated analyzers and by manual microscopy using a KOVA chamber. Within-run coefficients of variation (CVs) for the control materials were 7.0-8.8% and 1.7-5.7% for the cobas u 701 and UF-1000i systems, respectively. Between-run CVs were 8.5-9.8% and 2.7-5.4%, respectively. Both instruments showed good linearity and negligible carry-over. For red blood cells (RBC), white blood cells (WBC), and epithelial cells (EPI), the overall concordance rates within one grade of difference among the three methods were good (78.6-86.0%, 88.7-93.8%, and 81.3-90.7%, respectively). The concordance rate for casts was poor (66.5-68.9%). Compared with manual microscopy, the two automated sediment analyzers tested in this study showed satisfactory analytical performances for RBC, WBC, and EPI. However, for other urine sediment particles confirmation by visual microscopy is still required. © 2016 Wiley Periodicals, Inc.

Atomic Force Microscopy Based Cell Shape Index

NASA Astrophysics Data System (ADS)

Adia-Nimuwa, Usienemfon; Mujdat Tiryaki, Volkan; Hartz, Steven; Xie, Kan; Ayres, Virginia

2013-03-01

Stellation is a measure of cell physiology and pathology for several cell groups including neural, liver and pancreatic cells. In the present work, we compare the results of a conventional two-dimensional shape index study of both atomic force microscopy (AFM) and fluorescent microscopy images with the results obtained using a new three-dimensional AFM-based shape index similar to sphericity index. The stellation of astrocytes is investigated on nanofibrillar scaffolds composed of electrospun polyamide nanofibers that has demonstrated promise for central nervous system (CNS) repair. Recent work by our group has given us the ability to clearly segment the cells from nanofibrillar scaffolds in AFM images. The clear-featured AFM images indicated that the astrocyte processes were longer than previously identified at 24h. It was furthermore shown that cell spreading could vary significantly as a function of environmental parameters, and that AFM images could record these variations. The new three-dimensional AFM-based shape index incorporates the new information: longer stellate processes and cell spreading. The support of NSF PHY-095776 is acknowledged.

Surface plasmon holographic microscopy for near-field refractive index detection and thin film mapping

NASA Astrophysics Data System (ADS)

Zhao, Jianlin; Zhang, Jiwei; Dai, Siqing; Di, Jianglei; Xi, Teli

2018-02-01

Surface plasmon microscopy (SPM) is widely applied for label-free detection of changes of refractive index and concentration, as well as mapping thin films in near field. Traditionally, the SPM systems are based on the detection of light intensity or phase changes. Here, we present two kinds of surface plasmon holographic microscopy (SPHM) systems for amplitude- and phase-contrast imaging simultaneously. Through recording off-axis holograms and numerical reconstruction, the complex amplitude distributions of surface plasmon resonance (SPR) images can be obtained. According to the Fresnel's formula, in a prism/ gold/ dielectric structure, the reflection phase shift is uniquely decided by refractive index of the dielectric. By measuring the phase shift difference of the reflected light exploiting prism-coupling SPHM system based on common-path interference configuration, monitoring tiny refractive index variation and imaging biological tissue are performed. Furthermore, to characterize the thin film thickness in near field, we employ a four-layer SPR model in which the third film layer is within the evanescent field. The complex reflection coefficient, including the reflectivity and reflection phase shift, is uniquely decided by the film thickness. By measuring the complex amplitude distributions of the SPR images exploiting objective-coupling SPHM system based on common-path interference configuration, the thickness distributions of thin films are mapped with sub-nanometer resolution theoretically. Owing to its high temporal stability, the recommended SPHMs show great potentials for monitoring tiny refractive index variations, imaging biological tissues and mapping thin films in near field with dynamic, nondestructive and full-field measurement capabilities in chemistry, biomedicine field, etc.

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

PubMed Central

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

2017-01-01

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

Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples

NASA Astrophysics Data System (ADS)

Funane, Tsukasa; Hou, Steven S.; Zoltowska, Katarzyna Marta; van Veluw, Susanne J.; Berezovska, Oksana; Kumar, Anand T. N.; Bacskai, Brian J.

2018-05-01

We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.

In situ electrochemical digital holographic microscopy; a study of metal electrodeposition in deep eutectic solvents.

PubMed

Abbott, Andrew P; Azam, Muhammad; Ryder, Karl S; Saleem, Saima

2013-07-16

This study has shown for the first time that digital holographic microscopy (DHM) can be used as a new analytical tool in analysis of kinetic mechanism and growth during electrolytic deposition processes. Unlike many alternative established electrochemical microscopy methods such as probe microscopy, DHM is both the noninvasive and noncontact, the unique holographic imaging allows the observations and measurement to be made remotely. DHM also provides interferometric resolution (nanometer vertical scale) with a very short acquisition time. It is a surface metrology technique that enables the retrieval of information about a 3D structure from the phase contrast of a single hologram acquired using a conventional digital camera. Here DHM has been applied to investigate directly the electro-crystallization of a metal on a substrate in real time (in situ) from two deep eutectic solvent (DES) systems based on mixture of choline chloride and either urea or ethylene glycol. We show, using electrochemical DHM that the nucleation and growth of silver deposits in these systems are quite distinct and influenced strongly by the hydrogen bond donor of the DES.

Mapping optical path length and image enhancement using quantitative orientation-independent differential interference contrast microscopy

PubMed Central

Shribak, Michael; Larkin, Kieran G.; Biggs, David

2017-01-01

Abstract. We describe the principles of using orientation-independent differential interference contrast (OI-DIC) microscopy for mapping optical path length (OPL). Computation of the scalar two-dimensional OPL map is based on an experimentally received map of the OPL gradient vector field. Two methods of contrast enhancement for the OPL image, which reveal hardly visible structures and organelles, are presented. The results obtained can be used for reconstruction of a volume image. We have confirmed that a standard research grade light microscope equipped with the OI-DIC and 100×/1.3 NA objective lens, which was not specially selected for minimum wavefront and polarization aberrations, provides OPL noise level of ∼0.5  nm and lateral resolution if ∼300  nm at a wavelength of 546 nm. The new technology is the next step in the development of the DIC microscopy. It can replace standard DIC prisms on existing commercial microscope systems without modification. This will allow biological researchers that already have microscopy setups to expand the performance of their systems. PMID:28060991

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

PubMed

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

2017-08-01

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

Direct manipulation of metallic nanosheets by shear force microscopy.

PubMed

Bi, Z; Cai, W; Wang, Y; Shang, G

2018-05-15

Micro/nanomanipulation is a rapidly growing technology and holds promising applications in various fields, including photonic/electronic devices, chemical/biosensors etc. In this work, we present that shear force microscopy (ShFM) can be exploited to manipulate metallic nanosheets besides imaging. The manipulation is realized via controlling the shear force sensor probe position and shear force magnitude based on our homemade ShFM system under an optical microscopy for in situ observation. The main feature of the ShFM system is usage of a piezoelectric bimorph sensor, which has the ability of self-excitation and detection. Moreover, the shear force magnitude as a function of the spring constant of the sensor and setpoint is obtained, which indicates that operation modes can be switched between imaging and manipulation through designing the spring constant before experiment and changing the setpoint during manipulation process, respectively. We believe that this alternative manipulation technique could be used to assemble other nanostructures with different shapes, sizes and compositions for new properties and wider applications. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Miniature objective lens for array digital pathology: design improvement based on clinical evaluation

NASA Astrophysics Data System (ADS)

McCall, Brian; Pierce, Mark; Graviss, Edward A.; Richards-Kortum, Rebecca R.; Tkaczyk, Tomasz S.

2016-03-01

A miniature objective designed for digital detection of Mycobacterium tuberculosis (MTB) was evaluated for diagnostic accuracy. The objective was designed for array microscopy, but fabricated and evaluated at this stage of development as a single objective. The counts and diagnoses of patient samples were directly compared for digital detection and standard microscopy. The results were found to be correlated and highly concordant. The evaluation of this lens by direct comparison to standard fluorescence sputum smear microscopy presented unique challenges and led to some new insights in the role played by the system parameters of the microscope. The design parameters and how they were developed are reviewed in light of these results. New system parameters are proposed with the goal of easing the challenges of evaluating the miniature objective and maintaining the optical performance that produced the agreeable results presented without over-optimizing. A new design is presented that meets and exceeds these criteria.

Electrochemical force microscopy

DOEpatents

Kalinin, Sergei V.; Jesse, Stephen; Collins, Liam F.; Rodriguez, Brian J.

2017-01-10

A system and method for electrochemical force microscopy are provided. The system and method are based on a multidimensional detection scheme that is sensitive to forces experienced by a biased electrode in a solution. The multidimensional approach allows separation of fast processes, such as double layer charging, and charge relaxation, and slow processes, such as diffusion and faradaic reactions, as well as capturing the bias dependence of the response. The time-resolved and bias measurements can also allow probing both linear (small bias range) and non-linear (large bias range) electrochemical regimes and potentially the de-convolution of charge dynamics and diffusion processes from steric effects and electrochemical reactivity.

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo

PubMed Central

Chu, Jun; Oh, Young-Hee; Sens, Alex; Ataie, Niloufar; Dana, Hod; Macklin, John J.; Laviv, Tal; Welf, Erik S.; Dean, Kevin M.; Zhang, Feijie; Kim, Benjamin B.; Tang, Clement Tran; Hu, Michelle; Baird, Michelle A.; Davidson, Michael W.; Kay, Mark A.; Fiolka, Reto; Yasuda, Ryohei; Kim, Douglas S.; Ng, Ho-Leung; Lin, Michael Z.

2016-01-01

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals due to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright engineered orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins. PMID:27240196

Photo-actuating materials based on elastomers and modified carbon nanotubes

NASA Astrophysics Data System (ADS)

Czaniková, Klaudia; Krupa, Igor; Ilčíková, Markéta; Kasák, Peter; Chorvát, , Dušan; Valentin, Marian; Šlouf, Miroslav; Mosnáček, Jaroslav; Mičušík, Matej; Omastová, Mária

2012-01-01

The photo-actuating behavior of new polymeric nanocomposite materials based on a commercial elastomer, an ethylene-vinylacetate copolymer (EVA), filled with multiwalled carbon nanotubes (MWCNT) was investigated. A good dispersion of the MWCNT within the elastomeric matrix was ensured by using a novel, specific compatibilizer consisting of pyrenyl and cholesteryl groups. A uniaxial orientation of the MWCNT within the matrix was induced with shear forces by employing a special custom-made punch/die system. Good dispergation and alignment of the MWCNT within the matrix were demonstrated by scanning electron microscopy. Transmission electron microscopy showed a good dispersion of the MWCNT within the composite. Photo-actuation was qualitatively characterized by atomic force microscopy and quantitatively characterized by nanoindentation. The samples prepared in the form of Braille element showed expansion upon illumination by light diodes. The maximal height deformation changes about 15% was detected when a blue diode was used.

Quantification of transendothelial migration using three-dimensional confocal microscopy.

PubMed

Cain, Robert J; d'Água, Bárbara Borda; Ridley, Anne J

2011-01-01

Migration of cells across endothelial barriers, termed transendothelial migration (TEM), is an important cellular process that underpins the pathology of many disease states including chronic inflammation and cancer metastasis. While this process can be modeled in vitro using cultured cells, many model systems are unable to provide detailed visual information of cell morphologies and distribution of proteins such as junctional markers, as well as quantitative data on the rate of TEM. Improvements in imaging techniques have made microscopy-based assays an invaluable tool for studying this type of detailed cell movement in physiological processes. In this chapter, we describe a confocal microscopy-based method that can be used to assess TEM of both leukocytes and cancer cells across endothelial barriers in response to a chemotactic gradient, as well as providing information on their migration into a subendothelial extracellular matrix, designed to mimic that found in vivo.

Digital micromirror device-based laser-illumination Fourier ptychographic microscopy

PubMed Central

Kuang, Cuifang; Ma, Ye; Zhou, Renjie; Lee, Justin; Barbastathis, George; Dasari, Ramachandra R.; Yaqoob, Zahid; So, Peter T. C.

2015-01-01

We report a novel approach to Fourier ptychographic microscopy (FPM) by using a digital micromirror device (DMD) and a coherent laser source (532 nm) for generating spatially modulated sample illumination. Previously demonstrated FPM systems are all based on partially-coherent illumination, which offers limited throughput due to insufficient brightness. Our FPM employs a high power coherent laser source to enable shot-noise limited high-speed imaging. For the first time, a digital micromirror device (DMD), imaged onto the back focal plane of the illumination objective, is used to generate spatially modulated sample illumination field for ptychography. By coding the on/off states of the micromirrors, the illumination plane wave angle can be varied at speeds more than 4 kHz. A set of intensity images, resulting from different oblique illuminations, are used to numerically reconstruct one high-resolution image without obvious laser speckle. Experiments were conducted using a USAF resolution target and a fiber sample, demonstrating high-resolution imaging capability of our system. We envision that our approach, if combined with a coded-aperture compressive-sensing algorithm, will further improve the imaging speed in DMD-based FPM systems. PMID:26480361

Digital micromirror device-based laser-illumination Fourier ptychographic microscopy.

PubMed

Kuang, Cuifang; Ma, Ye; Zhou, Renjie; Lee, Justin; Barbastathis, George; Dasari, Ramachandra R; Yaqoob, Zahid; So, Peter T C

2015-10-19

We report a novel approach to Fourier ptychographic microscopy (FPM) by using a digital micromirror device (DMD) and a coherent laser source (532 nm) for generating spatially modulated sample illumination. Previously demonstrated FPM systems are all based on partially-coherent illumination, which offers limited throughput due to insufficient brightness. Our FPM employs a high power coherent laser source to enable shot-noise limited high-speed imaging. For the first time, a digital micromirror device (DMD), imaged onto the back focal plane of the illumination objective, is used to generate spatially modulated sample illumination field for ptychography. By coding the on/off states of the micromirrors, the illumination plane wave angle can be varied at speeds more than 4 kHz. A set of intensity images, resulting from different oblique illuminations, are used to numerically reconstruct one high-resolution image without obvious laser speckle. Experiments were conducted using a USAF resolution target and a fiber sample, demonstrating high-resolution imaging capability of our system. We envision that our approach, if combined with a coded-aperture compressive-sensing algorithm, will further improve the imaging speed in DMD-based FPM systems.

Multimodal optoacoustic and multiphoton fluorescence microscopy

NASA Astrophysics Data System (ADS)

Sela, Gali; Razansky, Daniel; Shoham, Shy

2013-03-01

Multiphoton microscopy is a powerful imaging modality that enables structural and functional imaging with cellular and sub-cellular resolution, deep within biological tissues. Yet, its main contrast mechanism relies on extrinsically administered fluorescent indicators. Here we developed a system for simultaneous multimodal optoacoustic and multiphoton fluorescence 3D imaging, which attains both absorption and fluorescence-based contrast by integrating an ultrasonic transducer into a two-photon laser scanning microscope. The system is readily shown to enable acquisition of multimodal microscopic images of fluorescently labeled targets and cell cultures as well as intrinsic absorption-based images of pigmented biological tissue. During initial experiments, it was further observed that that detected optoacoustically-induced response contains low frequency signal variations, presumably due to cavitation-mediated signal generation by the high repetition rate (80MHz) near IR femtosecond laser. The multimodal system may provide complementary structural and functional information to the fluorescently labeled tissue, by superimposing optoacoustic images of intrinsic tissue chromophores, such as melanin deposits, pigmentation, and hemoglobin or other extrinsic particle or dye-based markers highly absorptive in the NIR spectrum.

You can't measure what you can't see - detectors for microscopies

NASA Astrophysics Data System (ADS)

Denes, Peter

For centuries, the human eye has been the imaging detector of choice thanks to its high sensitivity, wide dynamic range, and direct connection to a built-in data recording and analysis system. The eye, however, is limited to visible light, which excludes microscopies with electrons and X-rays, and the built-in recording system stores archival information at very low rates. The former limitation has been overcome by ``indirect'' detectors, which convert probe particles to visible light, and the latter by a variety of recording techniques, from photographic film to semiconductor-based imagers. Semiconductor imagers have been used for decades as ``direct'' detectors in particle physics, and almost as long for hard X-rays. For soft X-ray microscopy, the challenge has been the small signal levels - plus getting the X-rays into the detector itself, given how quickly they are absorbed in inert layers. For electron microscopy, the challenge has been reconciling detector spatial resolution and pixel count with the large multiple scattering of electrons with energies used for microscopy. Further, a high recording rate (``movies'' rather than ``snapshots'') enables time-resolved studies, time-dependent corrections, shot-by-shot experiments and scanning techniques - at the expense of creating large data volumes. This talk will discuss solutions to these challenges, as well as an outlook towards future developments.

In vitro excystation of Echinostoma paraensei (Digenea: Echinostomatidae) metacercariae assessed by light microscopy, morphometry and confocal laser scanning microscopy.

PubMed

Souza, Joyce; Garcia, Juberlan; Neves, Renata H; Machado-Silva, José Roberto; Maldonado, Arnaldo

2013-12-01

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy. Copyright © 2013. Published by Elsevier Inc.

Microscopy using source and detector arrays

NASA Astrophysics Data System (ADS)

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

2016-03-01

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

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-08-01

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

Low-cost fluorescence microscopy for point-of-care cell imaging

NASA Astrophysics Data System (ADS)

Lochhead, Michael J.; Ives, Jeff; Givens, Monique; Delaney, Marie; Moll, Kevin; Myatt, Christopher J.

2010-02-01

Fluorescence microscopy has long been a standard tool in laboratory medicine. Implementation of fluorescence microscopy for near-patient diagnostics, however, has been limited due to cost and complexity associated with traditional fluorescence microscopy techniques. There is a particular need for robust, low-cost imaging in high disease burden areas in the developing world, where access to central laboratory facilities and trained staff is limited. Here we describe a point-of-care assay that combines a disposable plastic cartridge with an extremely low cost fluorescence imaging instrument. Based on a novel, multi-mode planar waveguide configuration, the system capitalizes on advances in volume-manufactured consumer electronic components to deliver an imaging system with minimal moving parts and low power requirements. A two-color cell imager is presented, with magnification optimized for enumeration of immunostained human T cells. To demonstrate the system, peripheral blood mononuclear cells were stained with fluorescently labeled anti-human-CD4 and anti-human-CD3 antibodies. Registered images were used to generate fractional CD4+ and CD3+ staining and enumeration results that show excellent correlation with flow cytometry. The cell imager is under development as a very low cost CD4+ T cell counter for HIV disease management in limited resource settings.

Design and Optimization of Nanomaterials for Sensing Applications

NASA Astrophysics Data System (ADS)

Sanderson, Robert Noboru

Nanomaterials, materials with one or more of their dimensions on the nanoscale, have emerged as an important field in the development of next-generation sensing systems. Their high surface-to-volume ratio makes them useful for sensing, but also makes them sensitive to processing defects and inherent material defects. To develop and optimize these systems, it is thus necessary to characterize these defects to understand their origin and how to work around them. Scanning probe microscopy (SPM) techniques like atomic force microscopy (AFM) and scanning tunneling microscopy (STM) are important characterization methods which can measure nanoscale topography and electronic structure. These methods are appealing in nanomaterial systems because they are non-damaging and provide local, high-resolution data, and so are capable of detecting nanoscale features such as single defect sites. There are difficulties, however, in the interpretation of SPM data. For instance, AFM-based methods are prone to experimental artifacts due to long-range interactions, such as capacitive crosstalk in Kelvin probe force microscopy (KPFM), and artifacts due to the finite size of the probe tip, such as incorrect surface tracking at steep topographical features. Mechanical characterization (via force spectroscopy) of nanomaterials with significant nanoscale variations, such as tethered lipid bilayer membranes (tLBMs), is also difficult since variations in the bulk system's mechanical behavior must be distinguished from local fluctuations. Additionally, interpretation of STM data is non-trivial due to local variations in electron density in addition to topographical variations. In this thesis we overcome some limitations of SPM methods by supplementing them with additional surface analytical methods as well as computational methods, and we characterize several nanomaterial systems. Current-carrying vapor-liquid-solid Si nanowires (useful for interdigitated-electrode-based sensors) are characterized using finite-element-method (FEM)-supplemented KPFM to retrieve useful information about processing defects, contact resistance, and the primary charge carriers. Next, a tLBM system's stiffness and the stiffness' dependence on tethering molecule concentration is measured using statistical analysis of thousands of AFM force spectra, demonstrating a biosensor-compatible system with a controllable bulk rigidity. Finally, we utilize surface analytical techniques to inform the development of a novel three-dimensional graphene system for sensing applications.

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.

Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy

PubMed Central

Rehberg, Markus; Krombach, Fritz; Pohl, Ulrich; Dietzel, Steffen

2011-01-01

Second and Third Harmonic Generation (SHG and THG) microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus) muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy. PMID:22140560

Laser scanning saturated structured illumination microscopy based on phase modulation

NASA Astrophysics Data System (ADS)

Huang, Yujia; Zhu, Dazhao; Jin, Luhong; Kuang, Cuifang; Xu, Yingke; Liu, Xu

2017-08-01

Wide-field saturated structured illumination microscopy has not been widely used due to the requirement of high laser power. We propose a novel method called laser scanning saturated structured illumination microscopy (LS-SSIM), which introduces high order of harmonics frequency and greatly reduces the required laser power for SSIM imaging. To accomplish that, an excitation PSF with two peaks is generated and scanned along different directions on the sample. Raw images are recorded cumulatively by a CCD detector and then reconstructed to form a high-resolution image with extended optical transfer function (OTF). Our theoretical analysis and simulation results show that LS-SSIM method reaches a resolution of 0.16 λ, equivalent to 2.7-fold resolution than conventional wide-field microscopy. In addition, LS-SSIM greatly improves the optical sectioning capability of conventional wide-field illumination system by diminishing our-of-focus light. Furthermore, this modality has the advantage of implementation in multi-photon microscopy with point scanning excitation to image samples in greater depths.

Analysis-Preserving Video Microscopy Compression via Correlation and Mathematical Morphology

PubMed Central

Shao, Chong; Zhong, Alfred; Cribb, Jeremy; Osborne, Lukas D.; O’Brien, E. Timothy; Superfine, Richard; Mayer-Patel, Ketan; Taylor, Russell M.

2015-01-01

The large amount video data produced by multi-channel, high-resolution microscopy system drives the need for a new high-performance domain-specific video compression technique. We describe a novel compression method for video microscopy data. The method is based on Pearson's correlation and mathematical morphology. The method makes use of the point-spread function (PSF) in the microscopy video acquisition phase. We compare our method to other lossless compression methods and to lossy JPEG, JPEG2000 and H.264 compression for various kinds of video microscopy data including fluorescence video and brightfield video. We find that for certain data sets, the new method compresses much better than lossless compression with no impact on analysis results. It achieved a best compressed size of 0.77% of the original size, 25× smaller than the best lossless technique (which yields 20% for the same video). The compressed size scales with the video's scientific data content. Further testing showed that existing lossy algorithms greatly impacted data analysis at similar compression sizes. PMID:26435032

Understanding materials challenges for rechargeable ion batteries with in situ transmission electron microscopy

NASA Astrophysics Data System (ADS)

Yuan, Yifei; Amine, Khalil; Lu, Jun; Shahbazian-Yassar, Reza

2017-08-01

An in-depth understanding of material behaviours under complex electrochemical environment is critical for the development of advanced materials for the next-generation rechargeable ion batteries. The dynamic conditions inside a working battery had not been intensively explored until the advent of various in situ characterization techniques. Real-time transmission electron microscopy of electrochemical reactions is one of the most significant breakthroughs poised to enable radical shift in our knowledge on how materials behave in the electrochemical environment. This review, therefore, summarizes the scientific discoveries enabled by in situ transmission electron microscopy, and specifically emphasizes the applicability of this technique to address the critical challenges in the rechargeable ion battery electrodes, electrolyte and their interfaces. New electrochemical systems such as lithium-oxygen, lithium-sulfur and sodium ion batteries are included, considering the rapidly increasing application of in situ transmission electron microscopy in these areas. A systematic comparison between lithium ion-based electrochemistry and sodium ion-based electrochemistry is also given in terms of their thermodynamic and kinetic differences. The effect of the electron beam on the validity of in situ observation is also covered. This review concludes by providing a renewed perspective for the future directions of in situ transmission electron microscopy in rechargeable ion batteries.

Understanding materials challenges for rechargeable ion batteries with in situ transmission electron microscopy

PubMed Central

Yuan, Yifei; Amine, Khalil; Lu, Jun; Shahbazian-Yassar, Reza

2017-01-01

An in-depth understanding of material behaviours under complex electrochemical environment is critical for the development of advanced materials for the next-generation rechargeable ion batteries. The dynamic conditions inside a working battery had not been intensively explored until the advent of various in situ characterization techniques. Real-time transmission electron microscopy of electrochemical reactions is one of the most significant breakthroughs poised to enable radical shift in our knowledge on how materials behave in the electrochemical environment. This review, therefore, summarizes the scientific discoveries enabled by in situ transmission electron microscopy, and specifically emphasizes the applicability of this technique to address the critical challenges in the rechargeable ion battery electrodes, electrolyte and their interfaces. New electrochemical systems such as lithium–oxygen, lithium–sulfur and sodium ion batteries are included, considering the rapidly increasing application of in situ transmission electron microscopy in these areas. A systematic comparison between lithium ion-based electrochemistry and sodium ion-based electrochemistry is also given in terms of their thermodynamic and kinetic differences. The effect of the electron beam on the validity of in situ observation is also covered. This review concludes by providing a renewed perspective for the future directions of in situ transmission electron microscopy in rechargeable ion batteries.

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

PubMed

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

2014-01-01

Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

Low vibration laboratory with a single-stage vibration isolation for microscopy applications.

PubMed

Voigtländer, Bert; Coenen, Peter; Cherepanov, Vasily; Borgens, Peter; Duden, Thomas; Tautz, F Stefan

2017-02-01

The construction and the vibrational performance of a low vibration laboratory for microscopy applications comprising a 100 ton floating foundation supported by passive pneumatic isolators (air springs), which rest themselves on a 200 ton solid base plate, are discussed. The optimization of the air spring system leads to a vibration level on the floating floor below that induced by an acceleration of 10 ng for most frequencies. Additional acoustic and electromagnetic isolation is accomplished by a room-in-room concept.

Simultaneous off-axis multiplexed holography and regular fluorescence microscopy of biological cells.

PubMed

Nygate, Yoav N; Singh, Gyanendra; Barnea, Itay; Shaked, Natan T

2018-06-01

We present a new technique for obtaining simultaneous multimodal quantitative phase and fluorescence microscopy of biological cells, providing both quantitative phase imaging and molecular specificity using a single camera. Our system is based on an interferometric multiplexing module, externally positioned at the exit of an optical microscope. In contrast to previous approaches, the presented technique allows conventional fluorescence imaging, rather than interferometric off-axis fluorescence imaging. We demonstrate the presented technique for imaging fluorescent beads and live biological cells.

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

NASA Astrophysics Data System (ADS)

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

2018-05-01

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

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

PubMed

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

2018-05-14

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

Computer-aided screening system for cervical precancerous cells based on field emission scanning electron microscopy and energy dispersive x-ray images and spectra

NASA Astrophysics Data System (ADS)

Jusman, Yessi; Ng, Siew-Cheok; Hasikin, Khairunnisa; Kurnia, Rahmadi; Osman, Noor Azuan Bin Abu; Teoh, Kean Hooi

2016-10-01

The capability of field emission scanning electron microscopy and energy dispersive x-ray spectroscopy (FE-SEM/EDX) to scan material structures at the microlevel and characterize the material with its elemental properties has inspired this research, which has developed an FE-SEM/EDX-based cervical cancer screening system. The developed computer-aided screening system consisted of two parts, which were the automatic features of extraction and classification. For the automatic features extraction algorithm, the image and spectra of cervical cells features extraction algorithm for extracting the discriminant features of FE-SEM/EDX data was introduced. The system automatically extracted two types of features based on FE-SEM/EDX images and FE-SEM/EDX spectra. Textural features were extracted from the FE-SEM/EDX image using a gray level co-occurrence matrix technique, while the FE-SEM/EDX spectra features were calculated based on peak heights and corrected area under the peaks using an algorithm. A discriminant analysis technique was employed to predict the cervical precancerous stage into three classes: normal, low-grade intraepithelial squamous lesion (LSIL), and high-grade intraepithelial squamous lesion (HSIL). The capability of the developed screening system was tested using 700 FE-SEM/EDX spectra (300 normal, 200 LSIL, and 200 HSIL cases). The accuracy, sensitivity, and specificity performances were 98.2%, 99.0%, and 98.0%, respectively.

Note: Design of FPGA based system identification module with application to atomic force microscopy

NASA Astrophysics Data System (ADS)

Ghosal, Sayan; Pradhan, Sourav; Salapaka, Murti

2018-05-01

The science of system identification is widely utilized in modeling input-output relationships of diverse systems. In this article, we report field programmable gate array (FPGA) based implementation of a real-time system identification algorithm which employs forgetting factors and bias compensation techniques. The FPGA module is employed to estimate the mechanical properties of surfaces of materials at the nano-scale with an atomic force microscope (AFM). The FPGA module is user friendly which can be interfaced with commercially available AFMs. Extensive simulation and experimental results validate the design.

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.

78 FR 33098 - Prospective Grant of Co-Exclusive Licenses: Multi-Focal Structured Illumination Microscopy...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-03

...-Exclusive Licenses: Multi-Focal Structured Illumination Microscopy Systems and Methods AGENCY: National... pertains to a system and method for digital confocal microscopy that rapidly processes enhanced images. In particular, the invention is a method for digital confocal microscopy that includes a digital mirror device...

Impact Ignition and Combustion Behavior of Amorphous Metal-Based Reactive Composites

NASA Astrophysics Data System (ADS)

Mason, Benjamin; Groven, Lori; Son, Steven

2013-06-01

Recently published molecular dynamic simulations have shown that metal-based reactive powder composites consisting of at least one amorphous component could lead to improved reaction performance due to amorphous materials having a zero heat of fusion, in addition to having high energy densities and potential uses such as structural energetic materials and enhanced blast materials. In order to investigate the feasibility of these systems, thermochemical equilibrium calculations were performed on various amorphous metal/metalloid based reactive systems with an emphasis on commercially available or easily manufactured amorphous metals, such as Zr and Ti based amorphous alloys in combination with carbon, boron, and aluminum. Based on the calculations and material availability material combinations were chosen. Initial materials were either mixed via a Resodyn mixer or mechanically activated using high energy ball milling where the microstructure of the milled material was characterized using x-ray diffraction, optical microscopy and scanning electron microscopy. The mechanical impact response and combustion behavior of select reactive systems was characterized using the Asay shear impact experiment where impact ignition thresholds, ignition delays, combustion velocities, and temperatures were quantified, and reported. Funding from the Defense Threat Reduction Agency (DTRA), Grant Number HDTRA1-10-1-0119. Counter-WMD basic research program, Dr. Suhithi M. Peiris, program director is gratefully acknowledged.

Modeling the depth-sectioning effect in reflection-mode dynamic speckle-field interferometric microscopy

PubMed Central

Zhou, Renjie; Jin, Di; Hosseini, Poorya; Singh, Vijay Raj; Kim, Yang-hyo; Kuang, Cuifang; Dasari, Ramachandra R.; Yaqoob, Zahid; So, Peter T. C.

2017-01-01

Unlike most optical coherence microscopy (OCM) systems, dynamic speckle-field interferometric microscopy (DSIM) achieves depth sectioning through the spatial-coherence gating effect. Under high numerical aperture (NA) speckle-field illumination, our previous experiments have demonstrated less than 1 μm depth resolution in reflection-mode DSIM, while doubling the diffraction limited resolution as under structured illumination. However, there has not been a physical model to rigorously describe the speckle imaging process, in particular explaining the sectioning effect under high illumination and imaging NA settings in DSIM. In this paper, we develop such a model based on the diffraction tomography theory and the speckle statistics. Using this model, we calculate the system response function, which is used to further obtain the depth resolution limit in reflection-mode DSIM. Theoretically calculated depth resolution limit is in an excellent agreement with experiment results. We envision that our physical model will not only help in understanding the imaging process in DSIM, but also enable better designing such systems for depth-resolved measurements in biological cells and tissues. PMID:28085800

Magnetoacoustic microscopic imaging of conductive objects and nanoparticles distribution

NASA Astrophysics Data System (ADS)

Liu, Siyu; Zhang, Ruochong; Luo, Yunqi; Zheng, Yuanjin

2017-09-01

Magnetoacoustic tomography has been demonstrated as a powerful and low-cost multi-wave imaging modality. However, due to limited spatial resolution and detection efficiency of magnetoacoustic signal, full potential of the magnetoacoustic imaging remains to be tapped. Here we report a high-resolution magnetoacoustic microscopy method, where magnetic stimulation is provided by a compact solenoid resonance coil connected with a matching network, and acoustic reception is realized by using a high-frequency focused ultrasound transducer. Scanning the magnetoacoustic microscopy system perpendicularly to the acoustic axis of the focused transducer would generate a two-dimensional microscopic image with acoustically determined lateral resolution. It is analyzed theoretically and demonstrated experimentally that magnetoacoustic generation in this microscopic system depends on the conductivity profile of conductive objects and localized distribution of superparamagnetic iron magnetic nanoparticles, based on two different but related implementations. The lateral resolution is characterized. Directional nature of magnetoacoustic vibration and imaging sensitivity for mapping magnetic nanoparticles are also discussed. The proposed microscopy system offers a high-resolution method that could potentially map intrinsic conductivity distribution in biological tissue and extraneous magnetic nanoparticles.

Microfluidics and Raman microscopy: current applications and future challenges.

PubMed

Chrimes, Adam F; Khoshmanesh, Khashayar; Stoddart, Paul R; Mitchell, Arnan; Kalantar-Zadeh, Kourosh

2013-07-07

Raman microscopy systems are becoming increasingly widespread and accessible for characterising chemical species. Microfluidic systems are also progressively finding their way into real world applications. Therefore, it is anticipated that the integration of Raman systems with microfluidics will become increasingly attractive and practical. This review aims to provide an overview of Raman microscopy-microfluidics integrated systems for researchers who are actively interested in utilising these tools. The fundamental principles and application strengths of Raman microscopy are discussed in the context of microfluidics. Various configurations of microfluidics that incorporate Raman microscopy methods are presented, with applications highlighted. Data analysis methods are discussed, with a focus on assisting the interpretation of Raman-microfluidics data from complex samples. Finally, possible future directions of Raman-microfluidic systems are presented.

Toward quantitative estimation of material properties with dynamic mode atomic force microscopy: a comparative study.

PubMed

Ghosal, Sayan; Gannepalli, Anil; Salapaka, Murti

2017-08-11

In this article, we explore methods that enable estimation of material properties with the dynamic mode atomic force microscopy suitable for soft matter investigation. The article presents the viewpoint of casting the system, comprising of a flexure probe interacting with the sample, as an equivalent cantilever system and compares a steady-state analysis based method with a recursive estimation technique for determining the parameters of the equivalent cantilever system in real time. The steady-state analysis of the equivalent cantilever model, which has been implicitly assumed in studies on material property determination, is validated analytically and experimentally. We show that the steady-state based technique yields results that quantitatively agree with the recursive method in the domain of its validity. The steady-state technique is considerably simpler to implement, however, slower compared to the recursive technique. The parameters of the equivalent system are utilized to interpret storage and dissipative properties of the sample. Finally, the article identifies key pitfalls that need to be avoided toward the quantitative estimation of material properties.

Automated 100-Position Specimen Loader and Image Acquisition System for Transmission Electron Microscopy

PubMed Central

Lefman, Jonathan; Morrison, Robert; Subramaniam, Sriram

2007-01-01

We report the development of a novel, multi-specimen imaging system for high-throughput transmission electron microscopy. Our cartridge-based loading system, called the “Gatling”, permits the sequential examination of as many as 100 specimens in the microscope for room temperature electron microscopy using mechanisms for rapid and automated specimen exchange. The software for the operation of the Gatling and automated data acquisition has been implemented in an updated version of our in-house program AutoEM. In the current implementation of the system, the time required to deliver 95 specimens into the microscope and collect overview images from each is about 13 hours. Regions of interest are identified from a low magnification atlas generation from each specimen and an unlimited number of higher magnifications images can be subsequently acquired from these regions using fully automated data acquisition procedures that can be controlled from a remote interface. We anticipate that the availability of the Gatling will greatly accelerate the speed of data acquisition for a variety of applications in biology, materials science and nanotechnology that require rapid screening and image analysis of multiple specimens. PMID:17240161

Preparation strategy and illumination of three-dimensional cell cultures in light sheet-based fluorescence microscopy

NASA Astrophysics Data System (ADS)

Bruns, Thomas; Schickinger, Sarah; Wittig, Rainer; Schneckenburger, Herbert

2012-10-01

A device for selective plane illumination microscopy (SPIM) of three-dimensional multicellular spheroids, in culture medium under stationary or microfluidic conditions, is described. Cell spheroids are located in a micro-capillary and a light sheet, for illumination, is generated in an optical setup adapted to a conventional inverse microscope. Layers of the sample, of about 10 μm or less in diameter, are, thus, illuminated selectively and imaged by high resolution fluorescence microscopy. SPIM is operated at low light exposure even if a larger number of layers is imaged and is easily combined with laser scanning microscopy. Chinese hamster ovary cells expressing a membrane-associated green fluorescent protein are used for preliminary tests, and the uptake of the fluorescent marker, acridine orange via a microfluidic system, is visualized to demonstrate its potential in cancer research such as for the detection of cellular responses to anticancer drugs.

Imaging the beating heart in the mouse using intravital microscopy techniques

PubMed Central

Vinegoni, Claudio; Aguirre, Aaron D; Lee, Sungon; Weissleder, Ralph

2017-01-01

Real-time microscopic imaging of moving organs at single-cell resolution represents a major challenge in studying complex biology in living systems. Motion of the tissue from the cardiac and respiratory cycles severely limits intravital microscopy by compromising ultimate spatial and temporal imaging resolution. However, significant recent advances have enabled single-cell resolution imaging to be achieved in vivo. In this protocol, we describe experimental procedures for intravital microscopy based on a combination of thoracic surgery, tissue stabilizers and acquisition gating methods, which enable imaging at the single-cell level in the beating heart in the mouse. Setup of the model is typically completed in 1 h, which allows 2 h or more of continuous cardiac imaging. This protocol can be readily adapted for the imaging of other moving organs, and it will therefore broadly facilitate in vivo high-resolution microscopy studies. PMID:26492138

Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine

PubMed Central

Vielreicher, M.; Schürmann, S.; Detsch, R.; Schmidt, M. A.; Buttgereit, A.; Boccaccini, A.; Friedrich, O.

2013-01-01

This review focuses on modern nonlinear optical microscopy (NLOM) methods that are increasingly being used in the field of tissue engineering (TE) to image tissue non-invasively and without labelling in depths unreached by conventional microscopy techniques. With NLOM techniques, biomaterial matrices, cultured cells and their produced extracellular matrix may be visualized with high resolution. After introducing classical imaging methodologies such as µCT, MRI, optical coherence tomography, electron microscopy and conventional microscopy two-photon fluorescence (2-PF) and second harmonic generation (SHG) imaging are described in detail (principle, power, limitations) together with their most widely used TE applications. Besides our own cell encapsulation, cell printing and collagen scaffolding systems and their NLOM imaging the most current research articles will be reviewed. These cover imaging of autofluorescence and fluorescence-labelled tissue and biomaterial structures, SHG-based quantitative morphometry of collagen I and other proteins, imaging of vascularization and online monitoring techniques in TE. Finally, some insight is given into state-of-the-art three-photon-based imaging methods (e.g. coherent anti-Stokes Raman scattering, third harmonic generation). This review provides an overview of the powerful and constantly evolving field of multiphoton microscopy, which is a powerful and indispensable tool for the development of artificial tissues in regenerative medicine and which is likely to gain importance also as a means for general diagnostic medical imaging. PMID:23864499

Simulation-based Extraction of Key Material Parameters from Atomic Force Microscopy

NASA Astrophysics Data System (ADS)

Alsafi, Huseen; Peninngton, Gray

Models for the atomic force microscopy (AFM) tip and sample interaction contain numerous material parameters that are often poorly known. This is especially true when dealing with novel material systems or when imaging samples that are exposed to complicated interactions with the local environment. In this work we use Monte Carlo methods to extract sample material parameters from the experimental AFM analysis of a test sample. The parameterized theoretical model that we use is based on the Virtual Environment for Dynamic AFM (VEDA) [1]. The extracted material parameters are then compared with the accepted values for our test sample. Using this procedure, we suggest a method that can be used to successfully determine unknown material properties in novel and complicated material systems. We acknowledge Fisher Endowment Grant support from the Jess and Mildred Fisher College of Science and Mathematics,Towson University.

A dark mode in scanning thermal microscopy

NASA Astrophysics Data System (ADS)

Ramiandrisoa, Liana; Allard, Alexandre; Joumani, Youssef; Hay, Bruno; Gomés, Séverine

2017-12-01

The need for high lateral spatial resolution in thermal science using Scanning Thermal Microscopy (SThM) has pushed researchers to look for more and more tiny probes. SThM probes have consequently become more and more sensitive to the size effects that occur within the probe, the sample, and their interaction. Reducing the tip furthermore induces very small heat flux exchanged between the probe and the sample. The measurement of this flux, which is exploited to characterize the sample thermal properties, requires then an accurate thermal management of the probe-sample system and to reduce any phenomenon parasitic to this system. Classical experimental methodologies must then be constantly questioned to hope for relevant and interpretable results. In this paper, we demonstrate and estimate the influence of the laser of the optical force detection system used in the common SThM setup that is based on atomic-force microscopy equipment on SThM measurements. We highlight the bias induced by the overheating due to the laser illumination on the measurements performed by thermoresistive probes (palladium probe from Kelvin Nanotechnology). To face this issue, we propose a new experimental procedure based on a metrological approach of the measurement: a SThM "dark mode." The comparison with the classical procedure using the laser shows that errors between 14% and 37% can be reached on the experimental data exploited to determine the heat flux transferred from the hot probe to the sample.

Quantitative chemical imaging with background-free multiplex coherent anti-Stokes Raman scattering by dual-soliton Stokes pulses

PubMed Central

Chen, Kun; Wu, Tao; Wei, Haoyun; Zhou, Tian; Li, Yan

2016-01-01

Coherent anti-Stokes Raman microscopy (CARS) is a quantitative, chemically specific, and label-free optical imaging technique for studying inhomogeneous systems. However, the complicating influence of the nonresonant response on the CARS signal severely limits its sensitivity and specificity and especially limits the extent to which CARS microscopy has been used as a fully quantitative imaging technique. On the basis of spectral focusing mechanism, we establish a dual-soliton Stokes based CARS microspectroscopy and microscopy scheme capable of quantifying the spatial information of densities and chemical composition within inhomogeneous samples, using a single fiber laser. Dual-soliton Stokes scheme not only removes the nonresonant background but also allows robust acquisition of multiple characteristic vibrational frequencies. This all-fiber based laser source can cover the entire fingerprint (800-2200 cm−1) region with a spectral resolution of 15 cm−1. We demonstrate that quantitative degree determination of lipid-chain unsaturation in the fatty acids mixture can be achieved by the characterization of C = C stretching and CH2 deformation vibrations. For microscopy purposes, we show that the spatially inhomogeneous distribution of lipid droplets can be further quantitatively visualized using this quantified degree of lipid unsaturation in the acyl chain for contrast in the hyperspectral CARS images. The combination of compact excitation source and background-free capability to facilitate extraction of quantitative composition information with multiplex spectral peaks will enable wider applications of quantitative chemical imaging in studying biological and material systems. PMID:27867704

Rapid diagnostic tests for malaria at sites of varying transmission intensity in Uganda.

PubMed

Hopkins, Heidi; Bebell, Lisa; Kambale, Wilson; Dokomajilar, Christian; Rosenthal, Philip J; Dorsey, Grant

2008-02-15

In Africa, fever is often treated presumptively as malaria, resulting in misdiagnosis and the overuse of antimalarial drugs. Rapid diagnostic tests (RDTs) for malaria may allow improved fever management. We compared RDTs based on histidine-rich protein 2 (HRP2) and RDTs based on Plasmodium lactate dehydrogenase (pLDH) with expert microscopy and PCR-corrected microscopy for 7000 patients at sites of varying malaria transmission intensity across Uganda. When all sites were considered, the sensitivity of the HRP2-based test was 97% when compared with microscopy and 98% when corrected by PCR; the sensitivity of the pLDH-based test was 88% when compared with microscopy and 77% when corrected by PCR. The specificity of the HRP2-based test was 71% when compared with microscopy and 88% when corrected by PCR; the specificity of the pLDH-based test was 92% when compared with microscopy and >98% when corrected by PCR. Based on Plasmodium falciparum PCR-corrected microscopy, the positive predictive value (PPV) of the HRP2-based test was high (93%) at all but the site with the lowest transmission rate; the pLDH-based test and expert microscopy offered excellent PPVs (98%) for all sites. The negative predictive value (NPV) of the HRP2-based test was consistently high (>97%); in contrast, the NPV for the pLDH-based test dropped significantly (from 98% to 66%) as transmission intensity increased, and the NPV for expert microscopy decreased significantly (99% to 54%) because of increasing failure to detect subpatent parasitemia. Based on the high PPV and NPV, HRP2-based RDTs are likely to be the best diagnostic choice for areas with medium-to-high malaria transmission rates in Africa.

Hybrid-coded 3D structured illumination imaging with Bayesian estimation (Conference Presentation)

NASA Astrophysics Data System (ADS)

Chen, Hsi-Hsun; Luo, Yuan; Singh, Vijay R.

2016-03-01

Light induced fluorescent microscopy has long been developed to observe and understand the object at microscale, such as cellular sample. However, the transfer function of lense-based imaging system limits the resolution so that the fine and detailed structure of sample cannot be identified clearly. The techniques of resolution enhancement are fascinated to break the limit of resolution for objective given. In the past decades, the resolution enhancement imaging has been investigated through variety of strategies, including photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), stimulated emission depletion (STED), and structure illuminated microscopy (SIM). In those methods, only SIM can intrinsically improve the resolution limit for a system without taking the structure properties of object into account. In this paper, we develop a SIM associated with Bayesian estimation, furthermore, with optical sectioning capability rendered from HiLo processing, resulting the high resolution through 3D volume. This 3D SIM can provide the optical sectioning and resolution enhancement performance, and be robust to noise owing to the Data driven Bayesian estimation reconstruction proposed. For validating the 3D SIM, we show our simulation result of algorithm, and the experimental result demonstrating the 3D resolution enhancement.

Automated Analysis of siRNA Screens of Virus Infected Cells Based on Immunofluorescence Microscopy

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

We present an image analysis approach as part of a high-throughput microscopy screening system based on cell arrays for the identification of genes involved in Hepatitis C and Dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in cells, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behavior of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

Concept for room temperature single-spin tunneling force microscopy with atomic spatial resolution

NASA Astrophysics Data System (ADS)

Payne, Adam

A study of a force detected single-spin magnetic resonance measurement concept with atomic spatial resolution is presented. The method is based upon electrostatic force detection of spin-selection rule controlled single electron tunneling between two electrically isolated paramagnetic states. Single-spin magnetic resonance detection is possible by measuring the force detected tunneling charge noise on and off spin resonance. Simulation results of this charge noise, based upon physical models of the tunneling and spin physics, are directly compared to measured atomic force microscopy (AFM) system noise. The results show that the approach could provide single-spin measurement of electrically isolated defect states with atomic spatial resolution at room temperature.

Quantitative measurements of nanoscale permittivity and conductivity using tuning-fork-based microwave impedance microscopy

NASA Astrophysics Data System (ADS)

Wu, Xiaoyu; Hao, Zhenqi; Wu, Di; Zheng, Lu; Jiang, Zhanzhi; Ganesan, Vishal; Wang, Yayu; Lai, Keji

2018-04-01

We report quantitative measurements of nanoscale permittivity and conductivity using tuning-fork (TF) based microwave impedance microscopy (MIM). The system is operated under the driving amplitude modulation mode, which ensures satisfactory feedback stability on samples with rough surfaces. The demodulated MIM signals on a series of bulk dielectrics are in good agreement with results simulated by finite-element analysis. Using the TF-MIM, we have visualized the evolution of nanoscale conductance on back-gated MoS2 field effect transistors, and the results are consistent with the transport data. Our work suggests that quantitative analysis of mesoscopic electrical properties can be achieved by near-field microwave imaging with small distance modulation.

Multiple excitation nano-spot generation and confocal detection for far-field microscopy.

PubMed

Mondal, Partha Pratim

2010-03-01

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

Multiple excitation nano-spot generation and confocal detection for far-field microscopy

NASA Astrophysics Data System (ADS)

Mondal, Partha Pratim

2010-03-01

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

Proximal design for a multimodality endoscope with multiphoton microscopy, optical coherence microscopy and visual modalities

NASA Astrophysics Data System (ADS)

Kiekens, Kelli C.; Talarico, Olivia; Barton, Jennifer K.

2018-02-01

A multimodality endoscope system has been designed for early detection of ovarian cancer. Multiple illumination and detection systems must be integrated in a compact, stable, transportable configuration to meet the requirements of a clinical setting. The proximal configuration presented here supports visible light navigation with a large field of view and low resolution, high resolution multiphoton microscopy (MPM), and high resolution optical coherence microscopy (OCM). All modalities are integrated into a single optical system in the endoscope. The system requires two light sources: a green laser for visible light navigation and a compact fiber based femtosecond laser for MPM and OCM. Using an inline wavelength division multiplexer, the two sources are combined into a single mode fiber. To accomplish OCM, a fiber coupler is used to separate the femtosecond laser into a reference arm and signal arm. The reflected reference arm and the signal from the sample are interfered and wavelength separated by a reflection grating and detected using a linear array. The MPM signal is collimated and goes through a series of filters to separate the 2nd and 3rd harmonics as well as twophoton excitation florescence (2PEF) and 3PEF. Each signal is independently detected on a photo multiplier tube and amplified. The visible light is collected by multiple high numerical aperture fibers at the endoscope tip which are bundled into one SMA adapter at the proximal end and connected to a photodetector. This integrated system design is compact, efficient and meets both optical and mechanical requirements for clinical applications.

Nonlinear adaptive optics: aberration correction in three photon fluorescence microscopy for mouse brain imaging

NASA Astrophysics Data System (ADS)

Sinefeld, David; Paudel, Hari P.; Wang, Tianyu; Wang, Mengran; Ouzounov, Dimitre G.; Bifano, Thomas G.; Xu, Chris

2017-02-01

Multiphoton fluorescence microscopy is a well-established technique for deep-tissue imaging with subcellular resolution. Three-photon microscopy (3PM) when combined with long wavelength excitation was shown to allow deeper imaging than two-photon microscopy (2PM) in biological tissues, such as mouse brain, because out-of-focus background light can be further reduced due to the higher order nonlinear excitation. As was demonstrated in 2PM systems, imaging depth and resolution can be improved by aberration correction using adaptive optics (AO) techniques which are based on shaping the scanning beam using a spatial light modulator (SLM). In this way, it is possible to compensate for tissue low order aberration and to some extent, to compensate for tissue scattering. Here, we present a 3PM AO microscopy system for brain imaging. Soliton self-frequency shift is used to create a femtosecond source at 1675 nm and a microelectromechanical (MEMS) SLM serves as the wavefront shaping device. We perturb the 1020 segment SLM using a modified nonlinear version of three-point phase shifting interferometry. The nonlinearity of the fluorescence signal used for feedback ensures that the signal is increasing when the spot size decreases, allowing compensation of phase errors in an iterative optimization process without direct phase measurement. We compare the performance for different orders of nonlinear feedback, showing an exponential growth in signal improvement as the nonlinear order increases. We demonstrate the impact of the method by applying the 3PM AO system for in-vivo mouse brain imaging, showing improvement in signal at 1-mm depth inside the brain.

Physical properties of inulin and inulin-orange juice: physical characterization and technological application.

PubMed

Saavedra-Leos, M Z; Leyva-Porras, C; Martínez-Guerra, E; Pérez-García, S A; Aguilar-Martínez, J A; Álvarez-Salas, C

2014-05-25

In this work two systems based on a carbohydrate polymer were studied: inulin as model system and inulin-orange juice as complex system. Both system were stored at different water activity conditions and subsequently characterized. Water adsorption isotherms type II were fitted by the GAB model and the water monolayer content was determined for each system. From thermal analyzes it was found that at low water activities (aw) systems were fully amorphous. As aw increased, crystallinity was developed. This behavior was corroborated by X-ray diffraction. In the inulin-orange juice system, crystallization appears at lower water activity caused by the intensification of the chemical interaction of the low molecular weight species contained in orange juice. Glass transition temperature (Tg), determined by modulated differential scanning calorimeter, decreased with aw. As water is adsorbed, the physical appearance of samples changed which could be observed by optical microscopy and effectively related with the microstructure found by scanning electron microscopy. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

Determining the size of nanoparticles in the example of magnetic iron oxide core-shell systems

NASA Astrophysics Data System (ADS)

Jarzębski, Maciej; Kościński, Mikołaj; Białopiotrowicz, Tomasz

2017-08-01

The size of nanoparticles is one of the most important factors for their possible applications. Various techniques for the nanoparticle size characterization are available. In this paper selected techniques will be considered base on the prepared core-shell magnetite nanoparticles. Magnetite is one of the most investigated and developed magnetic material. It shows interesting magnetic properties which can be used for biomedical applications, such as drug delivery, hypothermia and also as a contrast agent. To reduce the toxic effects of Fe3O4, magnetic core was covered by dextran and gelatin. Moreover, the shell was doped by fluorescent dye for confocal microscopy investigation. The main investigation focused on the methods for particles size determination of modified magnetite nanoparticles prepared with different techniques. The size distribution were obtained by nanoparticle tracking analysis, dynamic light scattering and transmission electron microscopy. Furthermore, fluorescent correlation spectroscopy (FCS) and confocal microscopy were used to compare the results for particle size determination of core-shell systems.

A coarse-grained model for DNA origami.

PubMed

Reshetnikov, Roman V; Stolyarova, Anastasia V; Zalevsky, Arthur O; Panteleev, Dmitry Y; Pavlova, Galina V; Klinov, Dmitry V; Golovin, Andrey V; Protopopova, Anna D

2018-02-16

Modeling tools provide a valuable support for DNA origami design. However, current solutions have limited application for conformational analysis of the designs. In this work we present a tool for a thorough study of DNA origami structure and dynamics. The tool is based on a novel coarse-grained model dedicated to geometry optimization and conformational analysis of DNA origami. We explored the ability of the model to predict dynamic behavior, global shapes, and fine details of two single-layer systems designed in hexagonal and square lattices using atomic force microscopy, Förster resonance energy transfer spectroscopy, and all-atom molecular dynamic simulations for validation of the results. We also examined the performance of the model for multilayer systems by simulation of DNA origami with published cryo-electron microscopy and atomic force microscopy structures. A good agreement between the simulated and experimental data makes the model suitable for conformational analysis of DNA origami objects. The tool is available at http://vsb.fbb.msu.ru/cosm as a web-service and as a standalone version.

A coarse-grained model for DNA origami

PubMed Central

Stolyarova, Anastasia V; Zalevsky, Arthur O; Panteleev, Dmitry Y; Pavlova, Galina V; Klinov, Dmitry V; Golovin, Andrey V; Protopopova, Anna D

2018-01-01

Abstract Modeling tools provide a valuable support for DNA origami design. However, current solutions have limited application for conformational analysis of the designs. In this work we present a tool for a thorough study of DNA origami structure and dynamics. The tool is based on a novel coarse-grained model dedicated to geometry optimization and conformational analysis of DNA origami. We explored the ability of the model to predict dynamic behavior, global shapes, and fine details of two single-layer systems designed in hexagonal and square lattices using atomic force microscopy, Förster resonance energy transfer spectroscopy, and all-atom molecular dynamic simulations for validation of the results. We also examined the performance of the model for multilayer systems by simulation of DNA origami with published cryo-electron microscopy and atomic force microscopy structures. A good agreement between the simulated and experimental data makes the model suitable for conformational analysis of DNA origami objects. The tool is available at http://vsb.fbb.msu.ru/cosm as a web-service and as a standalone version. PMID:29267876

Computer Vision Malaria Diagnostic Systems-Progress and Prospects.

PubMed

Pollak, Joseph Joel; Houri-Yafin, Arnon; Salpeter, Seth J

2017-01-01

Accurate malaria diagnosis is critical to prevent malaria fatalities, curb overuse of antimalarial drugs, and promote appropriate management of other causes of fever. While several diagnostic tests exist, the need for a rapid and highly accurate malaria assay remains. Microscopy and rapid diagnostic tests are the main diagnostic modalities available, yet they can demonstrate poor performance and accuracy. Automated microscopy platforms have the potential to significantly improve and standardize malaria diagnosis. Based on image recognition and machine learning algorithms, these systems maintain the benefits of light microscopy and provide improvements such as quicker scanning time, greater scanning area, and increased consistency brought by automation. While these applications have been in development for over a decade, recently several commercial platforms have emerged. In this review, we discuss the most advanced computer vision malaria diagnostic technologies and investigate several of their features which are central to field use. Additionally, we discuss the technological and policy barriers to implementing these technologies in low-resource settings world-wide.

Mapping Ionic Currents and Reactivity on the Nanoscale: Electrochemical Strain Microscopy

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

Kalinin, S.V.

2010-10-19

Solid-state electrochemical processes in oxides underpin a broad spectrum of energy and information storage devices, ranging from Li-ion and Li-air batteries, to solid oxide fuel cells (SOFC) to electroresistive and memristive systems. These functionalities are controlled by the bias-driven diffusive and electromigration transport of mobile ionic species, as well as intricate a set of electrochemical and defect-controlled reactions at interfaces and in bulk. Despite the wealth of device-level and atomistic studies, little is known on the mesoscopic mechanisms of ion diffusion and electronic transport on the level of grain clusters, individual grains, and extended defects. The development of the capabilitymore » for probing ion transport on the nanometer scale is a key to deciphering complex interplay between structure, functionality, and performance in these systems. Here we introduce Electrochemical Strain Microscopy, a scanning probe microscopy technique based on strong strain-bias coupling in the systems in which local ion concentrations are changed by electrical fields. The imaging capability, as well as time- and voltage spectroscopies analogous to traditional current based electrochemical characterization methods are developed. The reversible intercalation of Li and mapping electrochemical activity in LiCoO2 is demonstrated, illustrating higher Li diffusivity at non-basal planes and grain boundaries. In Si-anode device structure, the direct mapping of Li diffusion at extended defects and evolution of Li-activity with charge state is explored. The electrical field-dependence of Li mobility is studied to determine the critical bias required for the onset of electrochemical transformation, allowing reaction and diffusion processes in the battery system to be separated at each location. Finally, the applicability of ESM for probing oxygen vacancy diffusion and oxygen reduction/evolution reactions is illustrated, and the high resolution ESM maps are correlated with aberration corrected scanning transmission electron microscopy imaging. The future potential for deciphering mechanisms of electrochemical transformations on an atomically-defined single-defect level is discussed.« less

Media Matter: The Effect of Medium of Presentation on Student's Recognition of Histopathology.

PubMed

Telang, Ajay; Jong, Nynke De; Dalen, Jan Van

2016-12-01

Pathology teaching has undergone transformation with the introduction of virtual microscopy as a teaching and learning tool. To assess if dental students can identify histopathology irrespective of the media of presentation and if the media affect student's oral pathology case based learning scores. The perception of students towards "hybrid" approach in teaching and learning histopathology in oral pathology was also assessed. A controlled experiment was conduc-ted on year 4 and year 5 dental student groups using a perfor-mance test and a questionnaire survey. A response rate of 81% was noted for the performance test as well as the questionnaire survey. Results show a significant effect of media on performance of students with virtual microscopy bringing out the best performance across all student groups in case based learning scenarios. The order of preference for media was found to be virtual microscopy followed by photomicrographs and light microscopy. However, 94% of students still prefer the present hybrid system for teaching and learning of oral pathology. The study shows that identification of histo-pathology by students is dependent on media and the type of media has a significant effect on the performance. Virtual microscopy is strongly perceived as a useful tool for learning which thus brings out the best performance, however; the hybrid approach still remains the most preferred approach for histopathology learning.

Inhomogeneity Based Characterization of Distribution Patterns on the Plasma Membrane

PubMed Central

Paparelli, Laura; Corthout, Nikky; Wakefield, Devin L.; Sannerud, Ragna; Jovanovic-Talisman, Tijana; Annaert, Wim; Munck, Sebastian

2016-01-01

Cell surface protein and lipid molecules are organized in various patterns: randomly, along gradients, or clustered when segregated into discrete micro- and nano-domains. Their distribution is tightly coupled to events such as polarization, endocytosis, and intracellular signaling, but challenging to quantify using traditional techniques. Here we present a novel approach to quantify the distribution of plasma membrane proteins and lipids. This approach describes spatial patterns in degrees of inhomogeneity and incorporates an intensity-based correction to analyze images with a wide range of resolutions; we have termed it Quantitative Analysis of the Spatial distributions in Images using Mosaic segmentation and Dual parameter Optimization in Histograms (QuASIMoDOH). We tested its applicability using simulated microscopy images and images acquired by widefield microscopy, total internal reflection microscopy, structured illumination microscopy, and photoactivated localization microscopy. We validated QuASIMoDOH, successfully quantifying the distribution of protein and lipid molecules detected with several labeling techniques, in different cell model systems. We also used this method to characterize the reorganization of cell surface lipids in response to disrupted endosomal trafficking and to detect dynamic changes in the global and local organization of epidermal growth factor receptors across the cell surface. Our findings demonstrate that QuASIMoDOH can be used to assess protein and lipid patterns, quantifying distribution changes and spatial reorganization at the cell surface. An ImageJ/Fiji plugin of this analysis tool is provided. PMID:27603951

3D structured illumination microscopy using an incoherent illumination system based on a Fresnel biprism

NASA Astrophysics Data System (ADS)

Shabani, H.; Doblas, A.; Saavedra, G.; Preza, C.

2018-02-01

Three-dimensional (3D) structured illumination (SI) patterns that include lateral and axial variations have attracted more attention recently as their use in fluorescence microscope enhances the 3D resolution of the native imaging system. 3D SI patterns have already been created by interfering three mutually-coherent waves using a diffraction grating or some electro-optical devices such as spatial light modulators. Here, an interesting approach to generate a 3D SI pattern of tunable modulation frequency is shown. Our proposed illumination system is based on the incoherent illumination of a Fresnel biprism using several equidistant linear sources (i.e., slits). Previously, we investigated and compared numerically this tunable SI microscopy (SIM) system with the one achieved with three-wave interference. In this contribution, we implement our proposed incoherent 3D SIM system of tunable-frequency in an open-setup. We evaluate the axial confinement of the illumination pattern obtained with this system by recording the SI pattern using a mirror sample and different number of slits and compare these data with simulation results. Moreover, we verify that with a higher number of slits used, the axial confinement of the pattern increases, and consequently, the system's optical sectioning capability improves.

Erratum: "Low vibration laboratory with a single-stage vibration isolation for microscopy applications" [Rev. Sci. Instrum. 88, 023703 (2017)

NASA Astrophysics Data System (ADS)

Voigtländer, Bert; Coenen, Peter; Cherepanov, Vasily; Borgens, Peter; Duden, Thomas; Tautz, F. Stefan

2018-01-01

The construction and the vibrational performance of a low vibration laboratory for microscopy applications comprising a 100 ton floating foundation supported by passive pneumatic isolators (air springs), which rest themselves on a 200 ton solid base plate is discussed. The optimization of the air spring system lead to a vibration level on the floating floor below that induced by an acceleration of 10 ng for most frequencies. Additional acoustic and electromagnetic isolation is accomplished by a room-in-room concept.

Simulating contrast inversion in atomic force microscopy imaging with real-space pseudopotentials

NASA Astrophysics Data System (ADS)

Lee, Alex J.; Sakai, Yuki; Chelikowsky, James R.

2017-02-01

Atomic force microscopy (AFM) measurements have reported contrast inversions for systems such as Cu2N and graphene that can hamper image interpretation and characterization. Here, we apply a simulation method based on ab initio real-space pseudopotentials to gain an understanding of the tip-sample interactions that influence the inversion. We find that chemically reactive tips induce an attractive binding force that results in the contrast inversion. We find that the inversion is tip height dependent and not observed when using less reactive CO-functionalized tips.

Epidermis area detection for immunofluorescence microscopy

NASA Astrophysics Data System (ADS)

Dovganich, Andrey; Krylov, Andrey; Nasonov, Andrey; Makhneva, Natalia

2018-04-01

We propose a novel image segmentation method for immunofluorescence microscopy images of skin tissue for the diagnosis of various skin diseases. The segmentation is based on machine learning algorithms. The feature vector is filled by three groups of features: statistical features, Laws' texture energy measures and local binary patterns. The images are preprocessed for better learning. Different machine learning algorithms have been used and the best results have been obtained with random forest algorithm. We use the proposed method to detect the epidermis region as a part of pemphigus diagnosis system.

[Research on WiFi-based wireless microscopy on a mobile phone and its application].

PubMed

Hailan, Jin; Jing, Liu

2012-11-01

We proposed and realized a new device that acquires microscopic image wirelessly based on mobile phone and WiFi system. The mobile terminals could record, display and store the image from the far end via the wireless LAN. Using this system, a series of conceptual experiments on monitoring the microscopic images of common objects and liver cancer cells were successfully demonstrated. This system is expected to have important value in the experimental investigations on wirelessly monitoring the cell culture, and small insect etc.

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.

A Series of Zn(II) Terpyridine-Based Nitrate Complexes as Two-Photon Fluorescent Probe for Identifying Apoptotic and Living Cells via Subcellular Immigration.

PubMed

Liu, Dandan; Zhang, Mingzhu; Du, Wei; Hu, Lei; Li, Fei; Tian, Xiaohe; Wang, Aidong; Zhang, Qiong; Zhang, Zhongping; Wu, Jieying; Tian, Yupeng

2018-06-19

Two-photon active probe to label apoptotic cells plays a significant role in biological systems. However, discrimination of live/apoptotic cells at subcellular level under microscopy remains unachieved. Here, three novel Zn(II) terpyridine-based nitrate complexes (C1-C3) containing different pull/push units were designed. The structures of the ligands and their corresponding Zn(II) complexes were confirmed by single-crystal X-ray diffraction analysis. On the basis of the comprehensive comparison, C3 had a suitable two-photon absorption cross section in the near-infrared wavelength and good biocompatibility. Under two-photon confocal microscopy and transmission electron microscopy, it is found that C3 could target mitochondria in living cells but immigrate into the nucleolus during the apoptotic process. This dual-functional probe (C3) not only offers a valuable image tool but also acts as an indicator for cell mortality at subcellular level in a real-time manner.

Photothermal imaging of skeletal muscle mitochondria.

PubMed

Tomimatsu, Toru; Miyazaki, Jun; Kano, Yutaka; Kobayashi, Takayoshi

2017-06-01

The morphology and topology of mitochondria provide useful information about the physiological function of skeletal muscle. Previous studies of skeletal muscle mitochondria are based on observation with transmission, scanning electron microscopy or fluorescence microscopy. In contrast, photothermal (PT) microscopy has advantages over the above commonly used microscopic techniques because of no requirement for complex sample preparation by fixation or fluorescent-dye staining. Here, we employed the PT technique using a simple diode laser to visualize skeletal muscle mitochondria in unstained and stained tissues. The fine mitochondrial network structures in muscle fibers could be imaged with the PT imaging system, even in unstained tissues. PT imaging of tissues stained with toluidine blue revealed the structures of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria and the swelling behavior of mitochondria in damaged muscle fibers with sufficient image quality. PT image analyses based on fast Fourier transform (FFT) and Grey-level co-occurrence matrix (GLCM) were performed to derive the characteristic size of mitochondria and to discriminate the image patterns of normal and damaged fibers.

Analysis of Peroxisome Biogenesis in Pollen by Confocal Microscopy and Transmission Electron Microscopy.

PubMed

Jia, Peng-Fei; Li, Hong-Ju; Yang, Wei-Cai

2017-01-01

Peroxisome is an essential single-membrane bound organelle in most eukaryotic cells and functions in diverse cellular processes. De novo formation, division, and turnover of peroxisomes contribute to its biogenesis, morphology, and population regulation. In plants, peroxisome plays multiple roles, including metabolism, development, and stress response. Defective peroxisome biogenesis and development retard plant growth, adaption, and reproduction. Through tracing the subcellular localization of fluorescent reporter tagged matrix protein of peroxisome, fluorescence microscopy is a reliable and fast way to detect peroxisome biogenesis. Further fine-structural observation of peroxisome by TEM enables researchers to observe the detailed ultrastructure of its morphology and spatial contact with other organelles. Pollen grain is a specialized structure where two small sperm cells are enclosed in the cytoplasm of a large vegetative cell. Two features make pollen grain a good system to study peroxisome biogenesis: indispensable requirement of peroxisome for germination on the stigma and homogeneity. Here, we describe the methods of studying peroxisome biogenesis in Arabidopsis pollen grains by fluorescent live-imaging with confocal laser scanning microscopy (CLSM) and by DAB-staining based transmission electron microscopy (TEM).

Combined frequency modulated atomic force microscopy and scanning tunneling microscopy detection for multi-tip scanning probe microscopy applications

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

Morawski, Ireneusz; Institute of Experimental Physics, University of Wrocław, pl. M. Borna 9, 50-204 Wrocław; Spiegelberg, Richard

A method which allows scanning tunneling microscopy (STM) tip biasing independent of the sample bias during frequency modulated atomic force microscopy (AFM) operation is presented. The AFM sensor is supplied by an electronic circuit combining both a frequency shift signal and a tunneling current signal by means of an inductive coupling. This solution enables a control of the tip potential independent of the sample potential. Individual tip biasing is specifically important in order to implement multi-tip STM/AFM applications. An extensional quartz sensor (needle sensor) with a conductive tip is applied to record simultaneously topography and conductivity of the sample. Themore » high resonance frequency of the needle sensor (1 MHz) allows scanning of a large area of the surface being investigated in a reasonably short time. A recipe for the amplitude calibration which is based only on the frequency shift signal and does not require the tip being in contact is presented. Additionally, we show spectral measurements of the mechanical vibration noise of the scanning system used in the investigations.« less

Color calibration and fusion of lens-free and mobile-phone microscopy images for high-resolution and accurate color reproduction

NASA Astrophysics Data System (ADS)

Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

2016-06-01

Lens-free holographic microscopy can achieve wide-field imaging in a cost-effective and field-portable setup, making it a promising technique for point-of-care and telepathology applications. However, due to relatively narrow-band sources used in holographic microscopy, conventional colorization methods that use images reconstructed at discrete wavelengths, corresponding to e.g., red (R), green (G) and blue (B) channels, are subject to color artifacts. Furthermore, these existing RGB colorization methods do not match the chromatic perception of human vision. Here we present a high-color-fidelity and high-resolution imaging method, termed “digital color fusion microscopy” (DCFM), which fuses a holographic image acquired at a single wavelength with a color-calibrated image taken by a low-magnification lens-based microscope using a wavelet transform-based colorization method. We demonstrate accurate color reproduction of DCFM by imaging stained tissue sections. In particular we show that a lens-free holographic microscope in combination with a cost-effective mobile-phone-based microscope can generate color images of specimens, performing very close to a high numerical-aperture (NA) benchtop microscope that is corrected for color distortions and chromatic aberrations, also matching the chromatic response of human vision. This method can be useful for wide-field imaging needs in telepathology applications and in resource-limited settings, where whole-slide scanning microscopy systems are not available.

Color calibration and fusion of lens-free and mobile-phone microscopy images for high-resolution and accurate color reproduction

PubMed Central

Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

2016-01-01

Lens-free holographic microscopy can achieve wide-field imaging in a cost-effective and field-portable setup, making it a promising technique for point-of-care and telepathology applications. However, due to relatively narrow-band sources used in holographic microscopy, conventional colorization methods that use images reconstructed at discrete wavelengths, corresponding to e.g., red (R), green (G) and blue (B) channels, are subject to color artifacts. Furthermore, these existing RGB colorization methods do not match the chromatic perception of human vision. Here we present a high-color-fidelity and high-resolution imaging method, termed “digital color fusion microscopy” (DCFM), which fuses a holographic image acquired at a single wavelength with a color-calibrated image taken by a low-magnification lens-based microscope using a wavelet transform-based colorization method. We demonstrate accurate color reproduction of DCFM by imaging stained tissue sections. In particular we show that a lens-free holographic microscope in combination with a cost-effective mobile-phone-based microscope can generate color images of specimens, performing very close to a high numerical-aperture (NA) benchtop microscope that is corrected for color distortions and chromatic aberrations, also matching the chromatic response of human vision. This method can be useful for wide-field imaging needs in telepathology applications and in resource-limited settings, where whole-slide scanning microscopy systems are not available. PMID:27283459

Development of a HIV-1 Virus Detection System Based on Nanotechnology.

PubMed

Lee, Jin-Ho; Oh, Byung-Keun; Choi, Jeong-Woo

2015-04-27

Development of a sensitive and selective detection system for pathogenic viral agents is essential for medical healthcare from diagnostics to therapeutics. However, conventional detection systems are time consuming, resource-intensive and tedious to perform. Hence, the demand for sensitive and selective detection system for virus are highly increasing. To attain this aim, different aspects and techniques have been applied to develop virus sensor with improved sensitivity and selectivity. Here, among those aspects and techniques, this article reviews HIV virus particle detection systems incorporated with nanotechnology to enhance the sensitivity. This review mainly focused on four different detection system including vertically configured electrical detection based on scanning tunneling microscopy (STM), electrochemical detection based on direct electron transfer in virus, optical detection system based on localized surface plasmon resonance (LSPR) and surface enhanced Raman spectroscopy (SERS) using plasmonic nanoparticle.

Automated aberration correction of arbitrary laser modes in high numerical aperture systems.

PubMed

Hering, Julian; Waller, Erik H; Von Freymann, Georg

2016-12-12

Controlling the point-spread-function in three-dimensional laser lithography is crucial for fabricating structures with highest definition and resolution. In contrast to microscopy, aberrations have to be physically corrected prior to writing, to create well defined doughnut modes, bottlebeams or multi foci modes. We report on a modified Gerchberg-Saxton algorithm for spatial-light-modulator based automated aberration compensation to optimize arbitrary laser-modes in a high numerical aperture system. Using circularly polarized light for the measurement and first-guess initial conditions for amplitude and phase of the pupil function our scalar approach outperforms recent algorithms with vectorial corrections. Besides laser lithography also applications like optical tweezers and microscopy might benefit from the method presented.

Efficient Phase Unwrapping Architecture for Digital Holographic Microscopy

PubMed Central

Hwang, Wen-Jyi; Cheng, Shih-Chang; Cheng, Chau-Jern

2011-01-01

This paper presents a novel phase unwrapping architecture for accelerating the computational speed of digital holographic microscopy (DHM). A fast Fourier transform (FFT) based phase unwrapping algorithm providing a minimum squared error solution is adopted for hardware implementation because of its simplicity and robustness to noise. The proposed architecture is realized in a pipeline fashion to maximize throughput of the computation. Moreover, the number of hardware multipliers and dividers are minimized to reduce the hardware costs. The proposed architecture is used as a custom user logic in a system on programmable chip (SOPC) for physical performance measurement. Experimental results reveal that the proposed architecture is effective for expediting the computational speed while consuming low hardware resources for designing an embedded DHM system. PMID:22163688

Comparison of two structured illumination techniques based on different 3D illumination patterns

NASA Astrophysics Data System (ADS)

Shabani, H.; Patwary, N.; Doblas, A.; Saavedra, G.; Preza, C.

2017-02-01

Manipulating the excitation pattern in optical microscopy has led to several super-resolution techniques. Among different patterns, the lateral sinusoidal excitation was used for the first demonstration of structured illumination microscopy (SIM), which provides the fastest SIM acquisition system (based on the number of raw images required) compared to the multi-spot illumination approach. Moreover, 3D patterns that include lateral and axial variations in the illumination have attracted more attention recently as they address resolution enhancement in three dimensions. A threewave (3W) interference technique based on coherent illumination has already been shown to provide super-resolution and optical sectioning in 3D-SIM. In this paper, we investigate a novel tunable technique that creates a 3D pattern from a set of multiple incoherently illuminated parallel slits that act as light sources for a Fresnel biprism. This setup is able to modulate the illumination pattern in the object space both axially and laterally with adjustable modulation frequencies. The 3D forward model for the new system is developed here to consider the effect of the axial modulation due to the 3D patterned illumination. The performance of 3D-SIM based on 3W interference and the tunable system are investigated in simulation and compared based on two different criteria. First, restored images obtained for both 3D-SIM systems using a generalized Wiener filter are compared to determine the effect of the illumination pattern on the reconstruction. Second, the effective frequency response of both systems is studied to determine the axial and lateral resolution enhancement that is obtained in each case.

Identification of sandstone core damage using scanning electron microscopy

NASA Astrophysics Data System (ADS)

Ismail, Abdul Razak; Jaafar, Mohd Zaidi; Sulaiman, Wan Rosli Wan; Ismail, Issham; Shiunn, Ng Yinn

2017-12-01

Particles and fluids invasion into the pore spaces causes serious damage to the formation, resulting reduction in petroleum production. In order to prevent permeability damage for a well effectively, the damage mechanisms should be identified. In this study, water-based drilling fluid was compared to oil-based drilling fluids based on microscopic observation. The cores were damaged by several drilling fluid systems. Scanning electron microscope (SEM) was used to observe the damage mechanism caused by the drilling fluids. Results showed that the ester based drilling fluid system caused the most serious damage followed by synthetic oil based system and KCI-polymer system. Fine solids and filtrate migration and emulsion blockage are believed to be the major mechanisms controlling the changes in flow properties for the sandstone samples.

A fiber-optic-based imaging system for nondestructive assessment of cell-seeded tissue-engineered scaffolds.

PubMed

Hofmann, Matthias C; Whited, Bryce M; Criswell, Tracy; Rylander, Marissa Nichole; Rylander, Christopher G; Soker, Shay; Wang, Ge; Xu, Yong

2012-09-01

A major limitation in tissue engineering is the lack of nondestructive methods that assess the development of tissue scaffolds undergoing preconditioning in bioreactors. Due to significant optical scattering in most scaffolding materials, current microscope-based imaging methods cannot "see" through thick and optically opaque tissue constructs. To address this deficiency, we developed a fiber-optic-based imaging method that is capable of nondestructive imaging of fluorescently labeled cells through a thick and optically opaque scaffold, contained in a bioreactor. This imaging modality is based on the local excitation of fluorescent cells, the acquisition of fluorescence through the scaffold, and fluorescence mapping based on the position of the excitation light. To evaluate the capability and accuracy of the imaging system, human endothelial cells (ECs), stably expressing green fluorescent protein (GFP), were imaged through a fibrous scaffold. Without sacrificing the scaffolds, we nondestructively visualized the distribution of GFP-labeled cells through a ~500 μm thick scaffold with cell-level resolution and distinct localization. These results were similar to control images obtained using an optical microscope with direct line-of-sight access. Through a detailed quantitative analysis, we demonstrated that this method achieved a resolution on the order of 20-30 μm, with 10% or less deviation from standard optical microscopy. Furthermore, we demonstrated that the penetration depth of the imaging method exceeded that of confocal laser scanning microscopy by more than a factor of 2. Our imaging method also possesses a working distance (up to 8 cm) much longer than that of a standard confocal microscopy system, which can significantly facilitate bioreactor integration. This method will enable the nondestructive monitoring of ECs seeded on the lumen of a tissue-engineered vascular graft during preconditioning in vitro, as well as for other tissue-engineered constructs in the future.

Lipid-based liquid crystalline nanoparticles as oral drug delivery vehicles for poorly water-soluble drugs: cellular interaction and in vivo absorption

PubMed Central

Zeng, Ni; Gao, Xiaoling; Hu, Quanyin; Song, Qingxiang; Xia, Huimin; Liu, Zhongyang; Gu, Guangzhi; Jiang, Mengyin; Pang, Zhiqing; Chen, Hongzhuan; Chen, Jun; Fang, Liang

2012-01-01

Background Lipid-based liquid crystalline nanoparticles (LCNPs) have attracted growing interest as novel drug-delivery systems for improving the bioavailability of both hydrophilic and hydrophobic drugs. However, their cellular interaction and in vivo behavior have not been fully developed and characterized. Methods In this study, self-assembled LCNPs prepared from soy phosphatidylcholine and glycerol dioleate were developed as a platform for oral delivery of paclitaxel. The particle size of empty LCNPs and paclitaxel-loaded LCNPs was around 80 nm. The phase behavior of the liquid crystalline matrix was characterized using crossed polarized light microscopy and small-angle X-ray scattering, and showed both reversed cubic and hexagonal phase in the liquid crystalline matrix. Transmission electron microscopy and cryofield emission scanning electron microscopy analysis revealed an inner winding water channel in LCNPs and a “ ball-like”/“hexagonal” morphology. Results Cellular uptake of LCNPs in Caco-2 cells was found to be concentration-dependent and time-dependent, with involvement of both clathrin and caveolae/lipid raft-mediated endocytosis. Under confocal laser scanning microscopy, soy phosphatidylcholine was observed to segregate from the internalized LCNPs and to fuse with the cell membrane. An in vivo pharmacokinetic study showed that the oral bioavailability of paclitaxel-loaded LCNPs (13.16%) was 2.1 times that of Taxol® (the commercial formulation of paclitaxel, 6.39%). Conclusion The findings of this study suggest that this LCNP delivery system may be a promising candidate for improving the oral bioavailability of poorly water-soluble agents. PMID:22888230

Comparison of microscopy, ELISA, and real-time PCR for detection of Giardia intestinalis in human stool specimens

PubMed

Beyhan, Yunus Emre; Taş Cengiz, Zeynep

2017-08-23

Background/aim: This study included patients who had digestive system complaints between August 2015 and October 2015. The research was designed to compare conventional microscopy with an antigen detection ELISA kit and the TaqMan-based real-time PCR (RT-PCR) technique for detection of Giardia intestinalis in human stool specimens. Materials and methods: Samples were concentrated by formalin-ether sedimentation technique and microscopic examinations were carried out on wet mount slides. A commercially available ELISA kit (Giardia CELISA, Cellabs, Brookvale, Australia) was used for immunoassay. DNA was extracted from fecal samples of about 200 mg using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany) and the LightCycler Nano system (Roche Diagnostics, Mannheim, Germany) was used for the TaqMan-based RT-PCR assay. Results: A total of 94 stool samples, 38 of them diagnosed positive (40.4%) and 56 of them diagnosed negative by microscopy, were selected for evaluation by antigen detection and molecular assays. The prevalence of G. intestinalis infection was found as 46.8% (n: 44) and 79.8% (n: 75) by ELISA and RT-PCR, respectively. RT-PCR revealed by far the highest positivity rate compared to the other two methods. The difference between these methods was found to be statistically significant (P < 0.05). In comparison to PCR, the sensitivity and specificity of microscopy and ELISA were 50.7% and 100% and 53.3% and 79%, respectively. Conclusion: RT-PCR seems to be much more sensitive and beneficial for rapid and accurate diagnosis of G. intestinalis in human stools.

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

PubMed

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

2017-09-01

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

Atomic force microscopy based nanoindentation study of onion abaxial epidermis walls in aqueous environment

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

Xi, Xiaoning; Tittmann, Bernhard; Kim, Seong H.

An atomic force microscopy based nanoindentation method was employed to study how the structure of cellulose microfibril packing and matrix polymers affect elastic modulus of fully hydrated primary plant cell walls. The isolated, single-layered abaxial epidermis cell wall of an onion bulb was used as a test system since the cellulose microfibril packing in this cell wall is known to vary systematically from inside to outside scales and the most abundant matrix polymer, pectin, can easily be altered through simple chemical treatments such as ethylenediaminetetraacetic acid and calcium ions. Experimental results showed that the pectin network variation has significant impactsmore » on the cell wall modulus, and not the cellulose microfibril packing.« less

Moving towards the magnetoelectric graphene transistor

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

Cao, Shi; Xiao, Zhiyong; Kwan, Chun -Pui

Here, the interfacial charge transfer between mechanically exfoliated few-layer graphene and Cr 2O 3 (0001) surfaces has been investigated. Electrostatic force microscopy and Kelvin probe force microscopy studies point to hole doping of few-layer graphene, with up to a 150 meV shift in the Fermi level, an aspect that is confirmed by Raman spectroscopy. Density functional theory calculations furthermore confirm the p-type nature of the graphene/chromia interface and suggest that the chromia is able to induce a significant carrier spin polarization in the graphene layer. A large magnetoelectrically controlled magneto-resistance can therefore be anticipated in transistor structures based on thismore » system, a finding important for developing graphene-based spintronic applications.« less

Quantitative measurements of nanoscale permittivity and conductivity using tuning-fork-based microwave impedance microscopy

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

Wu, Xiaoyu; Hao, Zhenqi; Wu, Di

Here, we report quantitative measurements of nanoscale permittivity and conductivity using tuning-fork (TF) based microwave impedance microscopy (MIM). The system is operated under the driving amplitude modulation mode, which ensures satisfactory feedback stability on samples with rough surfaces. The demodulated MIM signals on a series of bulk dielectrics are in good agreement with results simulated by finite-element analysis. Using the TF-MIM, we have visualized the evolution of nanoscale conductance on back-gated MoS 2 field effect transistors, and the results are consistent with the transport data. Our work suggests that quantitative analysis of mesoscopic electrical properties can be achieved by near-fieldmore » microwave imaging with small distance modulation.« less

Quantitative measurements of nanoscale permittivity and conductivity using tuning-fork-based microwave impedance microscopy

DOE PAGES

Wu, Xiaoyu; Hao, Zhenqi; Wu, Di; ...

2018-04-01

Here, we report quantitative measurements of nanoscale permittivity and conductivity using tuning-fork (TF) based microwave impedance microscopy (MIM). The system is operated under the driving amplitude modulation mode, which ensures satisfactory feedback stability on samples with rough surfaces. The demodulated MIM signals on a series of bulk dielectrics are in good agreement with results simulated by finite-element analysis. Using the TF-MIM, we have visualized the evolution of nanoscale conductance on back-gated MoS 2 field effect transistors, and the results are consistent with the transport data. Our work suggests that quantitative analysis of mesoscopic electrical properties can be achieved by near-fieldmore » microwave imaging with small distance modulation.« less

Examination of biogenic selenium-containing nanosystems based on polyelectrolyte complexes by atomic force, Kelvin probe force and electron microscopy methods

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

Sukhanova, T. E., E-mail: tat-sukhanova@mail.ru; Vylegzhanina, M. E.; Valueva, S. V.

The morphology and electrical properties of biogenic selenium-containing nanosystems based on polyelectrolyte complexes (PECs) were examined using AFM, Kelvin Probe Force and electron microscopy methods. It has been found, that prepared nanostructures significantly differed in their morphological types and parameters. In particular, multilayers capsules can be produced via varying synthesis conditions, especially, the selenium–PEC mass ratio ν. At the “special point” (ν = 0.1), filled and hollow nano- and microcapsules are formed in the system. The multilayer character of the capsules walls is visible in the phase images. Kelvin Probe Force images showed the inhomogeneity of potential distribution in capsulesmore » and outside them.« less

Comparison of dislocation content measured with transmission electron microscopy and micro-Laue diffraction based streak analysis

DOE PAGES

Zhang, C.; Balachandran, S.; Eisenlohr, P.; ...

2017-10-04

The subsurface dislocation content in a Ti-5Al-2.5Sn (wt%) uniaxial tension sample deformed at ambient temperature was characterized by peak streak analysis of micro-Laue diffraction patterns collected non-destructively by differential aperture X-raymicroscopy, and with focused ion beam transmission electron microscopy of material in the same volume. This comparison reveals that micro-Laue diffraction streak analysis based on an edge dislocation assumption can accurately identify the dominant dislocation slip system history (Burgers vector and plane observed by TEM), despite the fact that dislocations have predominantly screw character. As a result, other dislocations identified by TEM were not convincingly discernible from the peak streakmore » analysis.« less

In-Process Atomic-Force Microscopy (AFM) Based Inspection

PubMed Central

Mekid, Samir

2017-01-01

A new in-process atomic-force microscopy (AFM) based inspection is presented for nanolithography to compensate for any deviation such as instantaneous degradation of the lithography probe tip. Traditional method used the AFM probes for lithography work and retract to inspect the obtained feature but this practice degrades the probe tip shape and hence, affects the measurement quality. This paper suggests a second dedicated lithography probe that is positioned back-to-back to the AFM probe under two synchronized controllers to correct any deviation in the process compared to specifications. This method shows that the quality improvement of the nanomachining, in progress probe tip wear, and better understanding of nanomachining. The system is hosted in a recently developed nanomanipulator for educational and research purposes. PMID:28561747

Moving towards the magnetoelectric graphene transistor

DOE PAGES

Cao, Shi; Xiao, Zhiyong; Kwan, Chun -Pui; ...

2017-10-30

Here, the interfacial charge transfer between mechanically exfoliated few-layer graphene and Cr 2O 3 (0001) surfaces has been investigated. Electrostatic force microscopy and Kelvin probe force microscopy studies point to hole doping of few-layer graphene, with up to a 150 meV shift in the Fermi level, an aspect that is confirmed by Raman spectroscopy. Density functional theory calculations furthermore confirm the p-type nature of the graphene/chromia interface and suggest that the chromia is able to induce a significant carrier spin polarization in the graphene layer. A large magnetoelectrically controlled magneto-resistance can therefore be anticipated in transistor structures based on thismore » system, a finding important for developing graphene-based spintronic applications.« less

Comparison of dislocation content measured with transmission electron microscopy and micro-Laue diffraction based streak analysis

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

Zhang, C.; Balachandran, S.; Eisenlohr, P.

The subsurface dislocation content in a Ti-5Al-2.5Sn (wt%) uniaxial tension sample deformed at ambient temperature was characterized by peak streak analysis of micro-Laue diffraction patterns collected non-destructively by differential aperture X-raymicroscopy, and with focused ion beam transmission electron microscopy of material in the same volume. This comparison reveals that micro-Laue diffraction streak analysis based on an edge dislocation assumption can accurately identify the dominant dislocation slip system history (Burgers vector and plane observed by TEM), despite the fact that dislocations have predominantly screw character. As a result, other dislocations identified by TEM were not convincingly discernible from the peak streakmore » analysis.« less

A GPU-based computer-assisted microscopy system for assessing the importance of different families of histological characteristics in cancer diagnosis

NASA Astrophysics Data System (ADS)

Glotsos, Dimitris; Kostopoulos, Spiros; Sidiropoulos, Konstantinos; Ravazoula, Panagiota; Kalatzis, Ioannis; Asvestas, Pantelis; Cavouras, Dionisis

2014-01-01

In this study a Computer-Aided Microscopy (CAM) system is proposed for investigating the importance of the histological criteria involved in diagnosing of cancers in microscopy in order to suggest the more informative features for discriminating low from high-grade brain tumours. Four families of criteria have been examined, involving the greylevel variations (i.e. texture), the morphology (i.e. roundness), the architecture (i.e. cellularity) and the overall tumour qualities (expert's ordinal scale). The proposed CAM system was constructed using a modified Seeded Region Growing algorithm for image segmentation, and the Probabilistic Neural Network classifier for image classification. The implementation was designed on a commercial Graphics Processing Unit card using parallel programming. The system's performance using textural, morphological, architectural and ordinal information was 90.8%, 87.0%, 81.2% and 88.9% respectively. Results indicate that nuclei texture is the most important family of features regarding the degree of malignancy, and, thus, may guide more accurate predictions for discriminating low from high grade gliomas. Considering that nuclei texture is almost impractical to be encoded by visual observation, the need to incorporate computer-aided diagnostic tools as second opinion in daily clinical practice of diagnosing rare brain tumours may be justified.

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

NASA Astrophysics Data System (ADS)

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

2015-03-01

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

Development of eddy current microscopy for high resolution electrical conductivity imaging using atomic force microscopy.

PubMed

Nalladega, V; Sathish, S; Jata, K V; Blodgett, M P

2008-07-01

We present a high resolution electrical conductivity imaging technique based on the principles of eddy current and atomic force microscopy (AFM). An electromagnetic coil is used to generate eddy currents in an electrically conducting material. The eddy currents generated in the conducting sample are detected and measured with a magnetic tip attached to a flexible cantilever of an AFM. The eddy current generation and its interaction with the magnetic tip cantilever are theoretically modeled using monopole approximation. The model is used to estimate the eddy current force between the magnetic tip and the electrically conducting sample. The theoretical model is also used to choose a magnetic tip-cantilever system with appropriate magnetic field and spring constant to facilitate the design of a high resolution electrical conductivity imaging system. The force between the tip and the sample due to eddy currents is measured as a function of the separation distance and compared to the model in a single crystal copper. Images of electrical conductivity variations in a polycrystalline dual phase titanium alloy (Ti-6Al-4V) sample are obtained by scanning the magnetic tip-cantilever held at a standoff distance from the sample surface. The contrast in the image is explained based on the electrical conductivity and eddy current force between the magnetic tip and the sample. The spatial resolution of the eddy current imaging system is determined by imaging carbon nanofibers in a polymer matrix. The advantages, limitations, and applications of the technique are discussed.

Scanning electrochemical microscopy based evaluation of influence of pH on bioelectrochemical activity of yeast cells - Saccharomyces cerevisiae.

PubMed

Ramanavicius, A; Morkvenaite-Vilkonciene, I; Kisieliute, A; Petroniene, J; Ramanaviciene, A

2017-01-01

In this research scanning electrochemical microscopy was applied for the investigation of immobilized yeast Saccharomyces cerevisiae cells. Two redox mediators based system was applied in order to increase the efficiency of charge transfer from yeast cells. 9,10-phenanthrenequinone (PQ) was applied as a lipophilic redox mediator, which has the ability to cross the cell's membrane; another redox mediator was ferricyanide, which acted as a hydrophylic electron acceptor able to transfer electrons from the PQ to the working electrode of SECM. Hill's function was applied to determine the optimal pH for this described SECM-based system. The influence of pH on cell viability could be well described by Hill's function. It was determined that at pH 6.5 the PQ has a minimal toxic influence on yeast cells, and the kinetics of metabolic processes in cells as well as electron transfer rate achieved in consecutive action of both redox mediators were appropriate to achieve optimal current signals. Copyright © 2016 Elsevier B.V. All rights reserved.

An automated real-time microscopy system for analysis of fluorescence resonance energy transfer

NASA Astrophysics Data System (ADS)

Bernardini, André; Wotzlaw, Christoph; Lipinski, Hans-Gerd; Fandrey, Joachim

2010-05-01

Molecular imaging based on Fluorescence Resonance Energy Transfer (FRET) is widely used in cellular physiology both for protein-protein interaction analysis and detecting conformational changes of single proteins, e.g. during activation of signaling cascades. However, getting reliable results from FRET measurements is still hampered by methodological problems such as spectral bleed through, chromatic aberration, focal plane shifts and false positive FRET. Particularly false positive FRET signals caused by random interaction of the fluorescent dyes can easily lead to misinterpretation of the data. This work introduces a Nipkow Disc based FRET microscopy system, that is easy to operate without expert knowledge of FRET. The system automatically accounts for all relevant sources of errors and provides various result presentations of two, three and four dimensional FRET data. Two examples are given to demonstrate the scope of application. An interaction analysis of the two subunits of the hypoxia-inducible transcription factor 1 demonstrates the use of the system as a tool for protein-protein interaction analysis. As an example for time lapse observations, the conformational change of the fluorophore labeled heat shock protein 33 in the presence of oxidant stress is shown.

Accuracy of an Immunochromatographic Diagnostic Test (ICT Malaria Combo Cassette Test) Compared to Microscopy among under Five-Year-Old Children when Diagnosing Malaria in Equatorial Guinea

PubMed Central

Portero, José-Luis; Rubio-Yuste, Maria; Descalzo, Miguel Angel; Raso, Jose; Lwanga, Magdalena; Obono, Jaquelina; Nseng, Gloria; Benito, Agustin; Cano, Jorge

2010-01-01

Conventional malaria diagnosis based on microscopy raises serious difficulties in weak health systems. Cost-effective and sensitive rapid diagnostic tests have been recently proposed as alternatives to microscopy. In Equatorial Guinea, a study was conducted to assess the reliability of a rapid diagnostic test compared to microscopy. The study was designed in accordance with the directives of the Standards for Reporting Diagnostic Accuracy Initiative (STARD). Peripheral thick and thin films for the microscopy diagnosis and a rapid immunochromatographic test (ICT Malaria Combo Cassette Test) were performed on under five-year-old children with malaria suspicion. The ICT test detected Plasmodium spp. infection with a sensitivity of 81.5% and a specificity of 81.9% while P. falciparum diagnosis occurred with a sensitivity of 69.7% and a specificity of 73.7%. The sensitivity of the ICT test increased with higher parasitemias. The general results showed little concordance between the ICT test and microscopy (kappa = 0.28, se: 0.04). In Equatorial Guinea, the ICT Malaria Combo Cassette Test has proven to be an acceptable test to detect high P. falciparum parasitemias. However, the decrease of sensitivity at medium and low parasitemias hampers that ICT can replace properly performed microscopy at present in the diagnosis of malaria in children. PMID:22332024

Diatom Valve Three-Dimensional Representation: A New Imaging Method Based on Combined Microscopies

PubMed Central

Ferrara, Maria Antonietta; De Tommasi, Edoardo; Coppola, Giuseppe; De Stefano, Luca; Rea, Ilaria; Dardano, Principia

2016-01-01

The frustule of diatoms, unicellular microalgae, shows very interesting photonic features, generally related to its complicated and quasi-periodic micro- and nano-structure. In order to simulate light propagation inside and through this natural structure, it is important to develop three-dimensional (3D) models for synthetic replica with high spatial resolution. In this paper, we present a new method that generates images of microscopic diatoms with high definition, by merging scanning electron microscopy and digital holography microscopy or atomic force microscopy data. Starting from two digital images, both acquired separately with standard characterization procedures, a high spatial resolution (Δz = λ/20, Δx = Δy ≅ 100 nm, at least) 3D model of the object has been generated. Then, the two sets of data have been processed by matrix formalism, using an original mathematical algorithm implemented on a commercially available software. The developed methodology could be also of broad interest in the design and fabrication of micro-opto-electro-mechanical systems. PMID:27690008

Three-dimensional molecular mapping of a multiple emulsion by means of CARS microscopy.

PubMed

Meyer, Tobias; Akimov, Denis; Tarcea, Nicolae; Chatzipapadopoulos, Susana; Muschiolik, Gerald; Kobow, Jens; Schmitt, Michael; Popp, Jürgen

2008-02-07

Multiple emulsions consisting of water droplets dispersed in an oil phase containing emulsifier which is emulsified in an outer water phase (W/O/W) are of great interest in pharmacology for developing new drugs, in the nutrition sciences for designing functional food, and in biology as model systems for cell organelles such as liposomes. In the food industry multiple emulsions with high sugar content in the aqueous phase can be used for the production of sweets, because the high sugar content prevents deterioration. However, for these emulsions the refractive indexes of oil and aqueous phase are very similar. This seriously impedes the analysis of these emulsions, e.g., for process monitoring, because microscopic techniques based on transmission or reflection do not provide sufficient contrast. We have characterized the inner dispersed phase of concentrated W/O/W emulsions with the same refractive index of the three phases by micro Raman spectroscopy and investigated the composition and molecular distribution in water-oil-water emulsions by means of three-dimensional laser scanning CARS (coherent anti-Stokes Raman scattering) microscopy. CARS microscopy has been used to study water droplets dispersed in oil droplets at different Raman resonances to visualize different molecular species. Water droplets with a diameter of about 700 nm could clearly be visualized. The advantages of CARS microscopy for studying this particular system are emphasized by comparing this microscopic technique with conventional confocal reflection and transmission microscopies.

Developing methods based on light sheet fluorescence microscopy for biophysical investigations of larval zebrafish

NASA Astrophysics Data System (ADS)

Taormina, Michael J.

Adapting the tools of optical microscopy to the large-scale dynamic systems encountered in the development of multicellular organisms provides a path toward understanding the physical processes necessary for complex life to form and function. Obtaining quantitatively meaningful results from such systems has been challenging due to difficulty spanning the spatial and temporal scales representative of the whole, while also observing the many individual members from which complex and collective behavior emerges. A three-dimensional imaging technique known as light sheet fluorescence microscopy provides a number of significant benefits for surmounting these challenges and studying developmental systems. A thin plane of fluorescence excitation light is produced such that it coincides with the focal plane of an imaging system, providing rapid acquisition of optically sectioned images that can be used to construct a three-dimensional rendition of a sample. I discuss the implementation of this technique for use in larva of the model vertebrate Danio rerio (zebrafish). The nature of light sheet imaging makes it especially well suited to the study of large systems while maintaining good spatial resolution and minimizing damage to the specimen from excessive exposure to excitation light. I show the results from a comparative study that demonstrates the ability to image certain developmental processes non-destructively, while in contrast confocal microscopy results in abnormal growth due to phototoxicity. I develop the application of light sheet microscopy to the study of a previously inaccessible system: the bacterial colonization of a host organism. Using the technique, we are able to obtain a survey of the intestinal tract of a larval zebrafish and observe the location of microbes as they grow and establish a stable population in an initially germ free fish. Finally, I describe a new technique to measure the fluid viscosity of this intestinal environment in vivo using magnetically driven particles. By imaging such particles as they are oscillated in a frequency chirped field, it is possible to calculate properties such as the viscosity of the material in which they are embedded. Here I provide the first known measurement of intestinal mucus rheology in vivo.

High-Contrast Imaging of Cholesterol Crystals in Rabbit Arteries Ex Vivo Using LED-Based Polarization Microscopy.

PubMed

Cho, Seonghee; Kim, Kyungmin; Kim, Taehoon; Park, Hyoeun; Kim, Jin-Moo; Lee, SeungHoon; Kang, YeonSu; Chang, Kiyuk; Kim, Chulhong

2018-04-19

Detection of cholesterol crystals (Chcs) in atherosclerosis disease is important for understanding the pathophysiology of atherosclerosis. Polarization microscopy (PM) has been in use traditionally for detecting Chcs, but they have difficulty in distinguishing Chcs with other crystalline materials in tissue, such as collagens. Thus, most studies using PM have been limited to studying cell-level samples. Although various methods have been proposed to detect Chcs with high specificity, most of them have low signal-to-noise ratios, a high system construction cost, and are difficult to operate due to a complex protocol. To address these problems, we have developed a simple and inexpensive universal serial bus (USB) PM system equipped with a 5700 K cool-white light-emitting diode (LED). In this system, Chcs are shown in a light blue color while collagen is shown in a yellow color. More importantly, the contrast between Chcs and collagens is improved by a factor of 2.3 under an aqueous condition in these PM images. These imaging results are well-matched with the ones acquired with two-photon microscopy (TPM). The system can visualize the features of atherosclerosis that cannot be visualized by the conventional hematoxylin and eosin and oil-red-o staining methods. Thus, we believe that this simple USB PM system can be widely used to identify Chcs in atherosclerosis.

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

Simple fiber-optic confocal microscopy with nanoscale depth resolution beyond the diffraction barrier.

PubMed

Ilev, Ilko; Waynant, Ronald; Gannot, Israel; Gandjbakhche, Amir

2007-09-01

A novel fiber-optic confocal approach for ultrahigh depth-resolution (

Design and evaluation of precise current integrator for scanning probe microscopy

NASA Astrophysics Data System (ADS)

Raczkowski, Kamil; Piasecki, Tomasz; Rudek, Maciej; Gotszalk, Teodor

2017-03-01

Several of the scanning probe microscopy (SPM) techniques, such as the scanning tunnelling microscopy (STM) or conductive atomic force microscopy (C-AFM), rely on precise measurements of current flowing between the investigated sample and the conductive nanoprobe. The parameters of current-to-voltage converter (CVC), which should detect current in the picompere range, are of utmost importance to those systems as they determine the microscopes’ measuring capabilities. That was the motivation for research on the precise current integrator (PCI), described in this paper, which could be used as the CVC in the C-AFM systems. The main design goal of the PCI was to provide a small and versatile device with the sub-picoampere level resolution with high dynamic range in the order of nanoamperes. The PCI was based on the integrating amplifier (Texas Instruments DDC112) paired with a STM32F4 microcontroller unit (MCU).The gain and bandwidth of the PCI might be easily changed by varying the integration time and the feedback capacitance. Depending on these parameters it was possible to obtain for example the 2.15 pA resolution at 688 nA range with 1 kHz bandwidth or 7.4 fA resolution at 0.98 nA range with 10 Hz bandwidth. The measurement of sinusoidal current with 28 fA amplitude was also presented. The PCI was integrated with the C-AFM system and used in the highly ordered pyrolytic graphite (HOPG) and graphene samples imaging.

Comparative feasibility of implementing rapid diagnostic test and microscopy for parasitological diagnosis of malaria in Uganda.

PubMed

Batwala, Vincent; Magnussen, Pascal; Nuwaha, Fred

2011-12-19

In Uganda, parasite-based diagnosis is recommended for every patient suspected to have malaria before prescribing anti-malarials. However, the majority of patients are still treated presumptively especially in low-level health units. The feasibility of implementing parasite-based diagnosis for uncomplicated malaria in rural health centres (HCs) was investigated with a view to recommending measures for scaling up the policy. Thirty HCs were randomized to implement parasite-based diagnosis based on rapid diagnostic tests [RDTs] (n = 10), blood microscopy (n = 10) and presumptive diagnosis (control arm) (n = 10). Feasibility was assessed by comparing the proportion of patients who received parasite-based diagnosis; with a positive malaria parasite-based diagnosis who received artemether-lumefantrine (AL); with a negative malaria parasite-based diagnosis who received AL; and patient waiting time. Clinicaltrials.gov: NCT00565071. 102, 087 outpatients were enrolled. Patients were more likely to be tested in the RDT 44, 565 (96.6%) than in microscopy arm 19, 545 (60.9%) [RR: 1.59]. RDTs reduced patient waiting time compared to microscopy and were more convenient to health workers and patients. Majority 23, 804 (99.7%) in presumptive arm were prescribed AL. All (100%) of patients who tested positive for malaria in RDT and microscopy arms were prescribed anti-malarials. Parasitological-based diagnosis significantly reduced AL prescription in RDT arm [RR: 0.62] and microscopy arm [RR: 0.72] compared to presumptive treatment. Among patients not tested in the two intervention arms, 12, 044 (96.1%) in microscopy and 965 (61.6%) in RDT arm were treated with AL [RR: 1.56]. Overall 10, 558 (29.4%) with negative results [5, 110 (23.4%) in RDT and 5, 448 (39.0%) in microscopy arms] were prescribed AL. It was more feasible to implement parasite-based diagnosis for malaria using RDT than with microscopy. A high proportion of patients with negative malaria results are still prescribed anti-malarials. There is need to increase access to parasite-based diagnosis where microscopy is used. In order to fully harness the benefits of parasitological confirmation of malaria, it is necessary to reduce the prescription of anti-malarials in negative patients.

The combination of scanning electron and scanning probe microscopy

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

Sapozhnikov, I. D.; Gorbenko, O. M., E-mail: gorolga64@gmail.com; Felshtyn, M. L.

2016-06-17

We suggest the SPM module to combine SEM and SPM methods for studying surfaces. The module is based on the original mechanical moving and scanning system. The examples of studies of the steel surface microstructure in both SEM and SPM modes are presented.

In situ electron microscopy of Braille microsystems: photo-actuation of ethylene vinyl acetate/carbon nanotube composites

NASA Astrophysics Data System (ADS)

Czaniková, Klaudia; Krupa, Igor; Račko, Dušan; Šmatko, Vasilij; Campo, Eva M.; Pavlova, Ewa; Omastová, Mária

2015-02-01

The development of new types of tactile displays based on the actuation of composite materials can aid the visually impaired. Micro/nano systems based on ethylene vinyl acetate (EVA) polymeric matrices enriched with multiwalled carbon nanotubes (MWCNT) can produce ensembles capable of light-induced actuation. In this report, we investigate two types of commercial EVA copolymers matrices containing 28 and 50 wt% vinyl-acetate (VA). Non-covalent modification of carbon nanotubes was achieved through a compatibilization technique that appends the pyrenenyl and cholesteryl groups on the carbon nanotubes (CNTs) surface. EVA/MWCNT nanocomposites were prepared by casting from a solution. These composites were shaped into Braille elements using molds. The deformation of the Braille element (BE) under light-emitting diode (LED) illumination was observed for the first time by in situ scanning electron microscopy (SEM). The superior actuation performance promoted by the EVA/MWCNT nanocomposites indicates that these materials will be useful in the future as light-driven micro/nano system actuators.

Myoanatomy of the velvet worm leg revealed by laboratory-based nanofocus X-ray source tomography.

PubMed

Müller, Mark; de Sena Oliveira, Ivo; Allner, Sebastian; Ferstl, Simone; Bidola, Pidassa; Mechlem, Korbinian; Fehringer, Andreas; Hehn, Lorenz; Dierolf, Martin; Achterhold, Klaus; Gleich, Bernhard; Hammel, Jörg U; Jahn, Henry; Mayer, Georg; Pfeiffer, Franz

2017-11-21

X-ray computed tomography (CT) is a powerful noninvasive technique for investigating the inner structure of objects and organisms. However, the resolution of laboratory CT systems is typically limited to the micrometer range. In this paper, we present a table-top nanoCT system in conjunction with standard processing tools that is able to routinely reach resolutions down to 100 nm without using X-ray optics. We demonstrate its potential for biological investigations by imaging a walking appendage of Euperipatoides rowelli , a representative of Onychophora-an invertebrate group pivotal for understanding animal evolution. Comparative analyses proved that the nanoCT can depict the external morphology of the limb with an image quality similar to scanning electron microscopy, while simultaneously visualizing internal muscular structures at higher resolutions than confocal laser scanning microscopy. The obtained nanoCT data revealed hitherto unknown aspects of the onychophoran limb musculature, enabling the 3D reconstruction of individual muscle fibers, which was previously impossible using any laboratory-based imaging technique.

Two-photon excitation fluorescence bioassays.

PubMed

Hänninen, Pekka; Soukka, Jori; Soini, Juhani T

2008-01-01

Application of two-photon excitation of fluorescence in microscopy is one of the major discoveries of the "renaissance" of light microscopy that started in the 1980s. The technique derives its advantages from the biologically "smooth" wavelength of the excitation light and the confinement of the excitation. Difficult, and seemingly nontransparent, samples may be imaged with the technique with good resolution. Although the bioresearch has been concentrating mostly on the positive properties of the technique for imaging, the same properties may be applied successfully to nonimaging bioassays. This article focuses on the development path of two-photon excitation-based assay system.

Visually testing the dynamic character of a blazed-angle adjustable grating by digital holographic microscopy.

PubMed

Qin, Chuan; Zhao, Jianlin; Di, Jianglei; Wang, Le; Yu, Yiting; Yuan, Weizheng

2009-02-10

We employed digital holographic microscopy to visually test microoptoelectromechanical systems (MOEMS). The sample is a blazed-angle adjustable grating. Considering the periodic structure of the sample, a local area unwrapping method based on a binary template was adopted to demodulate the fringes obtained by referring to a reference hologram. A series of holograms at different deformation states due to different drive voltages were captured to analyze the dynamic character of the MOEMS, and the uniformity of different microcantilever beams was also inspected. The results show this testing method is effective for a periodic structure.

A practical approach to the diagnosis of systemic amyloidoses.

PubMed

Fernández de Larrea, Carlos; Verga, Laura; Morbini, Patrizia; Klersy, Catherine; Lavatelli, Francesca; Foli, Andrea; Obici, Laura; Milani, Paolo; Capello, Gian Luca; Paulli, Marco; Palladini, Giovanni; Merlini, Giampaolo

2015-04-02

Accurate diagnosis of systemic amyloidosis is necessary both for assessing the prognosis and for delineating the appropriate treatment. It is based on histologic evidence of amyloid deposits and characterization of the amyloidogenic protein. We prospectively evaluated the diagnostic performance of immunoelectron microscopy (IEM) of abdominal fat aspirates from 745 consecutive patients with suspected systemic amyloidoses. All cases were extensively investigated with clinical and laboratory data, with a follow-up of at least 18 months. The 423 (56.8%) cases with confirmed systemic forms were used to estimate the diagnostic performance of IEM. Compared with Congo-red-based light microscopy, IEM was equally sensitive (75% to 80%) but significantly more specific (100% vs 80%; P < .001). In amyloid light-chain (AL) amyloidosis, κ cases were more difficult to diagnose (sensitivity 71%), whereas the analysis of abdominal aspirate was informative in only 40% of patients with transthyretin amyloidosis. We found a high prevalence (20%) of a monoclonal component in patients with non-AL amyloidosis, highlighting the risk of misdiagnosis and the need for unequivocal amyloid typing. Notably, IEM identified correctly the specific form of amyloidosis in >99% of the cases. IEM of abdominal fat aspirates is an effective tool in the routine diagnosis of systemic amyloidoses. © 2015 by The American Society of Hematology.

The Development of Teaching and Learning in Bright-Field Microscopy Technique

ERIC Educational Resources Information Center

Iskandar, Yulita Hanum P.; Mahmud, Nurul Ethika; Wahab, Wan Nor Amilah Wan Abdul; Jamil, Noor Izani Noor; Basir, Nurlida

2013-01-01

E-learning should be pedagogically-driven rather than technologically-driven. The objectives of this study are to develop an interactive learning system in bright-field microscopy technique in order to support students' achievement of their intended learning outcomes. An interactive learning system on bright-field microscopy technique was…

Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy

PubMed Central

Cha, Jae Won; Ballesta, Jerome; So, Peter T.C.

2010-01-01

The imaging depth of two-photon excitation fluorescence microscopy is partly limited by the inhomogeneity of the refractive index in biological specimens. This inhomogeneity results in a distortion of the wavefront of the excitation light. This wavefront distortion results in image resolution degradation and lower signal level. Using an adaptive optics system consisting of a Shack-Hartmann wavefront sensor and a deformable mirror, wavefront distortion can be measured and corrected. With adaptive optics compensation, we demonstrate that the resolution and signal level can be better preserved at greater imaging depth in a variety of ex-vivo tissue specimens including mouse tongue muscle, heart muscle, and brain. However, for these highly scattering tissues, we find signal degradation due to scattering to be a more dominant factor than aberration. PMID:20799824

Shack-Hartmann wavefront-sensor-based adaptive optics system for multiphoton microscopy.

PubMed

Cha, Jae Won; Ballesta, Jerome; So, Peter T C

2010-01-01

The imaging depth of two-photon excitation fluorescence microscopy is partly limited by the inhomogeneity of the refractive index in biological specimens. This inhomogeneity results in a distortion of the wavefront of the excitation light. This wavefront distortion results in image resolution degradation and lower signal level. Using an adaptive optics system consisting of a Shack-Hartmann wavefront sensor and a deformable mirror, wavefront distortion can be measured and corrected. With adaptive optics compensation, we demonstrate that the resolution and signal level can be better preserved at greater imaging depth in a variety of ex-vivo tissue specimens including mouse tongue muscle, heart muscle, and brain. However, for these highly scattering tissues, we find signal degradation due to scattering to be a more dominant factor than aberration.

Prototype of Kepler Processing Workflows For Microscopy And Neuroinformatics

PubMed Central

Astakhov, V.; Bandrowski, A.; Gupta, A.; Kulungowski, A.W.; Grethe, J.S.; Bouwer, J.; Molina, T.; Rowley, V.; Penticoff, S.; Terada, M.; Wong, W.; Hakozaki, H.; Kwon, O.; Martone, M.E.; Ellisman, M.

2016-01-01

We report on progress of employing the Kepler workflow engine to prototype “end-to-end” application integration workflows that concern data coming from microscopes deployed at the National Center for Microscopy Imaging Research (NCMIR). This system is built upon the mature code base of the Cell Centered Database (CCDB) and integrated rule-oriented data system (IRODS) for distributed storage. It provides integration with external projects such as the Whole Brain Catalog (WBC) and Neuroscience Information Framework (NIF), which benefit from NCMIR data. We also report on specific workflows which spawn from main workflows and perform data fusion and orchestration of Web services specific for the NIF project. This “Brain data flow” presents a user with categorized information about sources that have information on various brain regions. PMID:28479932

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.

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

PubMed

Ding, Qiuning; Tao, Chao; Liu, Xiaojun

2017-03-20

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

Analogous on-axis interference topographic phase microscopy (AOITPM).

PubMed

Xiu, P; Liu, Q; Zhou, X; Xu, Y; Kuang, C; Liu, X

2018-05-01

The refractive index (RI) of a sample as an endogenous contrast agent plays an important role in transparent live cell imaging. In tomographic phase microscopy (TPM), 3D quantitative RI maps can be reconstructed based on the measured projections of the RI in multiple directions. The resolution of the RI maps not only depends on the numerical aperture of the employed objective lens, but also is determined by the accuracy of the quantitative phase of the sample measured at multiple scanning illumination angles. This paper reports an analogous on-axis interference TPM, where the interference angle between the sample and reference beams is kept constant for projections in multiple directions to improve the accuracy of the phase maps and the resolution of RI tomograms. The system has been validated with both silica beads and red blood cells. Compared with conventional TPM, the proposed system acquires quantitative RI maps with higher resolution (420 nm @λ = 633 nm) and signal-to-noise ratio that can be beneficial for live cell imaging in biomedical applications. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Time-resolved fluorescence microscopy to study biologically related applications using sol-gel derived and cellular media

NASA Astrophysics Data System (ADS)

Toury, Marion; Chandler, Lin; Allison, Archie; Campbell, David; McLoskey, David; Holmes-Smith, A. Sheila; Hungerford, Graham

2011-03-01

Fluorescence microscopy provides a non-invasive means for visualising dynamic protein interactions. As well as allowing the calculation of kinetic processes via the use of time-resolved fluorescence, localisation of the protein within cells or model systems can be monitored. These fluorescence lifetime images (FLIM) have become the preferred technique for elucidating protein dynamics due to the fact that the fluorescence lifetime is an absolute measure, in the main independent of fluorophore concentration and intensity fluctuations caused by factors such as photobleaching. In this work we demonstrate the use of a time-resolved fluorescence microscopy, employing a high repetition rate laser excitation source applied to study the influence of a metal surface on fluorescence tagged protein and to elucidate viscosity using the fluorescence lifetime probe DASPMI. These were studied in a cellular environment (yeast) and in a model system based on a sol-gel derived material, in which silver nanostructures were formed in situ using irradiation from a semiconductor laser in CW mode incorporated on a compact time-resolved fluorescence microscope (HORIBA Scientific DeltaDiode and DynaMyc).

Screening photoswitching properties of synthesized BODIPY-based fluorophores for multispectral superresolution microscopy (MSSRM) (Conference Presentation)

NASA Astrophysics Data System (ADS)

Bittel, Amy M.; Saldivar, Isaac S.; Nan, Xiaolin; Gibbs, Summer L.

2016-02-01

Single-molecule localization microscopy (SMLM) utilizes photoswitchable fluorophores to detect biological entities with 10-20 nm resolution. Multispectral superresolution microscopy (MSSRM) extends SMLM functionality by improving its spectral resolution up to 5 fold facilitating imaging of multicomponent cellular structures or signaling pathways. Current commercial fluorophores are not ideal for MSSRM as they are not designed to photoswitch and do not adequately cover the visible and far-red spectral regions required for MSSRM imaging. To obtain optimal MSSRM spatial and spectral resolution, fluorophores with narrow emission spectra and controllable photoswitching properties are necessary. Herein, a library of BODIPY-based fluorophores was synthesized and characterized to create optimal photoswitchable fluorophores for MSSRM. BODIPY was chosen as the core structure as it is photostable, has high quantum yield, and controllable photoswitching. The BODIPY core was modified through the addition of various aromatic moieties, resulting in a spectrally diverse library. Photoswitching properties were characterized using a novel polyvinyl alcohol (PVA) based film methodology to isolate single molecules. The PVA film methodology enabled photoswitching assessment without the need for protein conjugation, greatly improving screening efficiency of the BODIPY library. Additionally, image buffer conditions were optimized for the BODIPY-based fluorophores through systematic testing of oxygen scavenger systems, redox components, and additives. Through screening the photoswitching properties of BODIPY-based compounds in PVA films with optimized imaging buffer we identified novel fluorophores well suited for SMLM and MSSRM.

Imaging latex–carbon nanotube composites by subsurface electrostatic force microscopy

DOE PAGES

Patel, Sajan; Petty, Clayton W.; Krafcik, Karen Lee; ...

2016-09-08

Electrostatic modes of atomic force microscopy have shown to be non-destructive and relatively simple methods for imaging conductors embedded in insulating polymers. Here we use electrostatic force microscopy to image the dispersion of carbon nanotubes in a latex-based conductive composite, which brings forth features not observed in previously studied systems employing linear polymer films. A fixed-potential model of the probe-nanotube electrostatics is presented which in principle gives access to the conductive nanoparticle's depth and radius, and the polymer film dielectric constant. Comparing this model to the data results in nanotube depths that appear to be slightly above the film–air interface.more » Furthermore, this result suggests that water-mediated charge build-up at the film–air interface may be the source of electrostatic phase contrast in ambient conditions.« less

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.

Voltage controlled nano-injection system for single-cell surgery

PubMed Central

Seger, R. Adam; Actis, Paolo; Penfold, Catherine; Maalouf, Michelle; Vilozny, Boaz; Pourmand, Nader

2015-01-01

Manipulation and analysis of single cells is the next frontier in understanding processes that control the function and fate of cells. Herein we describe a single-cell injection platform based on nanopipettes. The system uses scanning microscopy techniques to detect cell surfaces, and voltage pulses to deliver molecules into individual cells. As a proof of concept, we injected adherent mammalian cells with fluorescent dyes. PMID:22899383

Voltage controlled nano-injection system for single-cell surgery.

PubMed

Adam Seger, R; Actis, Paolo; Penfold, Catherine; Maalouf, Michelle; Vilozny, Boaz; Pourmand, Nader

2012-09-28

Manipulation and analysis of single cells is the next frontier in understanding processes that control the function and fate of cells. Herein we describe a single-cell injection platform based on nanopipettes. The system uses scanning microscopy techniques to detect cell surfaces, and voltage pulses to deliver molecules into individual cells. As a proof of concept, we injected adherent mammalian cells with fluorescent dyes.

Bioimaging with micro/nanoelectrode systems.

PubMed

Matsue, Tomokazu

2013-01-01

This article presents an overview of the recent progress made by our group in the development of bioelectrochemical imaging devices and systems with micro/nanoelectrodes. The topics include bioimaging of enzymes and live cells by scanning electrochemical microscopy (SECM), high-resolution bioimaging by SECM equipped with a nanoprobe, comprehensive measurements and bioimaging with local-redox cycling-based electrochemical (LRC-EC) devices, and rapid and sensitive bioimaging with BioLSI.

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

PubMed Central

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

2016-01-01

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

Super-resolution microscopy reveals protein spatial reorganization in early innate immune responses.

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

Carson, Bryan D.; Aaron, Jesse S.; Timlin, Jerilyn Ann

2010-10-01

Over the past decade optical approaches were introduced that effectively break the diffraction barrier. Of particular note were introductions of Stimulated Emission/Depletion (STED) microscopy, Photo-Activated Localization Microscopy (PALM), and the closely related Stochastic Optical Reconstruction Microscopy (STORM). STORM represents an attractive method for researchers, as it does not require highly specialized optical setups, can be implemented using commercially available dyes, and is more easily amenable to multicolor imaging. We implemented a simultaneous dual-color, direct-STORM imaging system through the use of an objective-based TIRF microscope and filter-based image splitter. This system allows for excitation and detection of two fluorophors simultaneously, viamore » projection of each fluorophor's signal onto separate regions of a detector. We imaged the sub-resolution organization of the TLR4 receptor, a key mediator of innate immune response, after challenge with lipopolysaccharide (LPS), a bacteria-specific antigen. While distinct forms of LPS have evolved among various bacteria, only some LPS variations (such as that derived from E. coli) typically result in significant cellular immune response. Others (such as from the plague bacteria Y. pestis) do not, despite affinity to TLR4. We will show that challenge with LPS antigens produces a statistically significant increase in TLR4 receptor clusters on the cell membrane, presumably due to recruitment of receptors to lipid rafts. These changes, however, are only detectable below the diffraction limit and are not evident using conventional imaging methods. Furthermore, we will compare the spatiotemporal behavior of TLR4 receptors in response to different LPS chemotypes in order to elucidate possible routes by which pathogens such as Y. pestis are able to circumvent the innate immune system. Finally, we will exploit the dual-color STORM capabilities to simultaneously image LPS and TLR4 receptors in the cellular membrane at resolutions at or below 40nm.« less

Optical Diagnostics in Medicine

NASA Astrophysics Data System (ADS)

Iftimia, Nicusor

2003-03-01

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

Photon-counting-based diffraction phase microscopy combined with single-pixel imaging

NASA Astrophysics Data System (ADS)

Shibuya, Kyuki; Araki, Hiroyuki; Iwata, Tetsuo

2018-04-01

We propose a photon-counting (PC)-based quantitative-phase imaging (QPI) method for use in diffraction phase microscopy (DPM) that is combined with a single-pixel imaging (SPI) scheme (PC-SPI-DPM). This combination of DPM with the SPI scheme overcomes a low optical throughput problem that has occasionally prevented us from obtaining quantitative-phase images in DPM through use of a high-sensitivity single-channel photodetector such as a photomultiplier tube (PMT). The introduction of a PMT allowed us to perform PC with ease and thus solved a dynamic range problem that was inherent to SPI. As a proof-of-principle experiment, we performed a comparison study of analogue-based SPI-DPM and PC-SPI-DPM for a 125-nm-thick indium tin oxide (ITO) layer coated on a silica glass substrate. We discuss the basic performance of the method and potential future modifications of the proposed system.

Hot Corrosion Behavior of Arc-Sprayed Highly Dense NiCr-Based Coatings in Chloride Salt Deposit

NASA Astrophysics Data System (ADS)

Qin, Enwei; Yin, Song; Ji, Hua; Huang, Qian; Liu, Zekun; Wu, Shuhui

2017-04-01

To make cities more environmentally friendly, combustible wastes tend to be incinerated in waste-to-energy power plant boilers. However, release of chlorine gas (Cl2) during incineration causes serious problems related to hot corrosion of boiler tubes and poses a safety threat for such plants. In this study, a pseudo-de Laval nozzle was employed in a twin-wire arc spray system to enhance the velocity of in-flight particles. Highly dense NiCr-based coatings were obtained using the modified nozzle gun. The coating morphology was characterized by optical microscopy and scanning electron microscopy, and hot corrosion testing was carried out in a synthetic molten chloride salt environment. Results showed that the dense NiCr-based coatings exhibited high resistance against corrosion by chlorine, which can be related to the typical splat lamellar microstructure and chemical composition as well as minor alloying elements such as Ti and Mo.

Systems microscopy: an emerging strategy for the life sciences.

PubMed

Lock, John G; Strömblad, Staffan

2010-05-01

Dynamic cellular processes occurring in time and space are fundamental to all physiology and disease. To understand complex and dynamic cellular processes therefore demands the capacity to record and integrate quantitative multiparametric data from the four spatiotemporal dimensions within which living cells self-organize, and to subsequently use these data for the mathematical modeling of cellular systems. To this end, a raft of complementary developments in automated fluorescence microscopy, cell microarray platforms, quantitative image analysis and data mining, combined with multivariate statistics and computational modeling, now coalesce to produce a new research strategy, "systems microscopy", which facilitates systems biology analyses of living cells. Systems microscopy provides the crucial capacities to simultaneously extract and interrogate multiparametric quantitative data at resolution levels ranging from the molecular to the cellular, thereby elucidating a more comprehensive and richly integrated understanding of complex and dynamic cellular systems. The unique capacities of systems microscopy suggest that it will become a vital cornerstone of systems biology, and here we describe the current status and future prospects of this emerging field, as well as outlining some of the key challenges that remain to be overcome. Copyright 2010 Elsevier Inc. All rights reserved.

Automated sample area definition for high-throughput microscopy.

PubMed

Zeder, M; Ellrott, A; Amann, R

2011-04-01

High-throughput screening platforms based on epifluorescence microscopy are powerful tools in a variety of scientific fields. Although some applications are based on imaging geometrically defined samples such as microtiter plates, multiwell slides, or spotted gene arrays, others need to cope with inhomogeneously located samples on glass slides. The analysis of microbial communities in aquatic systems by sample filtration on membrane filters followed by multiple fluorescent staining, or the investigation of tissue sections are examples. Therefore, we developed a strategy for flexible and fast definition of sample locations by the acquisition of whole slide overview images and automated sample recognition by image analysis. Our approach was tested on different microscopes and the computer programs are freely available (http://www.technobiology.ch). Copyright © 2011 International Society for Advancement of Cytometry.

Ultrafast, large-field multiphoton microscopy based on an acousto-optic deflector and a spatial light modulator.

PubMed

Shao, Yonghong; Qin, Wan; Liu, Honghai; Qu, Junle; Peng, Xiang; Niu, Hanben; Gao, Bruce Z

2012-07-01

We present an ultrafast, large-field multiphoton excitation fluorescence microscope with high lateral and axial resolutions based on a two-dimensional (2-D) acousto-optical deflector (AOD) scanner and spatial light modulator (SLM). When a phase-only SLM is used to shape the near-infrared light from a mode-locked titanium:sapphire laser into a multifocus array including the 0-order beam, a 136 μm × 136 μm field of view is achieved with a 60× objective using a 2-D AOD scanner without any mechanical scan element. The two-photon fluorescence image of a neuronal network that was obtained using this system demonstrates that our microscopy permits observation of dynamic biological events in a large field with high-temporal and -spatial resolution.

Multicell migration tracking within angiogenic networks by deep learning-based segmentation and augmented Bayesian filtering.

PubMed

Wang, Mengmeng; Ong, Lee-Ling Sharon; Dauwels, Justin; Asada, H Harry

2018-04-01

Cell migration is a key feature for living organisms. Image analysis tools are useful in studying cell migration in three-dimensional (3-D) in vitro environments. We consider angiogenic vessels formed in 3-D microfluidic devices (MFDs) and develop an image analysis system to extract cell behaviors from experimental phase-contrast microscopy image sequences. The proposed system initializes tracks with the end-point confocal nuclei coordinates. We apply convolutional neural networks to detect cell candidates and combine backward Kalman filtering with multiple hypothesis tracking to link the cell candidates at each time step. These hypotheses incorporate prior knowledge on vessel formation and cell proliferation rates. The association accuracy reaches 86.4% for the proposed algorithm, indicating that the proposed system is able to associate cells more accurately than existing approaches. Cell culture experiments in 3-D MFDs have shown considerable promise for improving biology research. The proposed system is expected to be a useful quantitative tool for potential microscopy problems of MFDs.

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.

Sensor Systems for Biological Agent Attacks: Protecting Buildings and Military Bases

DTIC Science & Technology

2004-01-01

simple aerosol detectors, to those that identify an agent based on its genetic, structural, or chemical properties , to so- called "functional...Cytometry, 122 Target Binding That Changes Detectable Properties of Smart Sensor Surfaces, 124 Colorimetric Detection, 124 Fluorescence Detection, 125 One...microscopy. In addition to particles directly derived from living organisms, other particles in air may also share properties with the bioaerosols

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

NASA Astrophysics Data System (ADS)

Guo, Kaikai; Zheng, Guoan

2017-03-01

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

Adaptive platform for fluorescence microscopy-based high-content screening

NASA Astrophysics Data System (ADS)

Geisbauer, Matthias; Röder, Thorsten; Chen, Yang; Knoll, Alois; Uhl, Rainer

2010-04-01

Fluorescence microscopy has become a widely used tool for the study of medically relevant intra- and intercellular processes. Extracting meaningful information out of a bulk of acquired images is usually performed during a separate post-processing task. Thus capturing raw data results in an unnecessary huge number of images, whereas usually only a few images really show the particular information that is searched for. Here we propose a novel automated high-content microscope system, which enables experiments to be carried out with only a minimum of human interaction. It facilitates a huge speed-increase for cell biology research and its applications compared to the widely performed workflows. Our fluorescence microscopy system can automatically execute application-dependent data processing algorithms during the actual experiment. They are used for image contrast enhancement, cell segmentation and/or cell property evaluation. On-the-fly retrieved information is used to reduce data and concomitantly control the experiment process in real-time. Resulting in a closed loop of perception and action the system can greatly decrease the amount of stored data on one hand and increases the relative valuable data content on the other hand. We demonstrate our approach by addressing the problem of automatically finding cells with a particular combination of labeled receptors and then selectively stimulate them with antagonists or agonists. The results are then compared against the results of traditional, static systems.

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.

Fracture behavior of nano-scale rubber-modified epoxies

NASA Astrophysics Data System (ADS)

Bacigalupo, Lauren N.

The primary focus of the first portion of this study is to compare physical and mechanical properties of a model epoxy that has been toughened with one of three different types of rubber-based modifier: a traditional telechelic oligomer (phase separates into micro-size particles), a core-shell latex particle (preformed nano-scale particles) and a triblock copolymer (self-assembles into nano-scale particles). The effect of modifier content on the physical properties of the matrix was determined using several thermal analysis methods, which provided insight into any inherent alterations of the epoxy matrix. Although the primary objective is to study the role of particle size on the fracture toughness, stiffness and strength were also determined since these properties are often reduced in rubber-toughened epoxies. It was found that since the CSR- and SBM-modified epoxies are composed of less rubber, thermal and mechanical properties of the epoxy were better maintained. In order to better understand the fracture behavior and mechanisms of the three types of rubber particles utilized in this study, extensive microscopy analysis was conducted. Scanning transmission electron microscopy (STEM) was used to quantify the volume fraction of particles, transmission optical microscopy (TOM) was used to determine plastic damage zone size, and scanning electron microscopy (SEM) was used to assess void growth in the plastic zone after fracture. By quantifying these characteristics, it was then possible to model the plastic damage zone size as well as the fracture toughness to elucidate the behavior of the rubber-modified epoxies. It was found that localized shear yielding and matrix void growth are the active toughening mechanisms in all rubber-modified epoxies in this study, however, matrix void growth was more prevalent. The second portion of this study investigated the use of three acrylate-based triblocks and four acrylate-based diblocks to modify a model epoxy system. By varying block lengths and the polarity of the epoxy-miscible blocks, a variety of morphologies were generated (such as spherical micelles, layer particles and worm-like micelles). It was found that in some cases, the epoxy-miscible block did not yield domains substantial enough to facilitate increases in toughness. Overall, the thermal and mechanical properties of the acrylate-based triblock- and diblock-modified epoxies were found to be similar to CTBN-modified epoxy, which was used as a control. However, there were properties that were improved with the acrylate-based diblock-modified epoxies when compared to the acrylate-based triblock modified epoxies. Specifically, the viscosity penalty of the diblock-modified epoxies was shown to be a marked improvement over the triblock-modified epoxies, especially given that the fracture toughness values are similar. This reduction in the viscosity penalty becomes an important criterion when considering processing procedures and applications. Additionally, comparing the morphology of the resulting modified-epoxies utilizing atomic force microscopy (AFM) and scanning electron microscopy (SEM) led to a better understanding of the relationship between the particle morphology obtained and the physical properties of the acrylate-based rubber-modified epoxy systems in this research.

sideSPIM – selective plane illumination based on a conventional inverted microscope

PubMed Central

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

2017-01-01

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

Plates-formes de microscopie et fluorescence par resonance de plasmons de surface appliquees a l'imagerie cellulaire

NASA Astrophysics Data System (ADS)

Chabot, Vincent

L'elaboration de nouveaux medicaments repose sur les etudes pharmacologiques, dont le role est d'identifier de nouveaux composes actifs ou de nouvelles cibles pharmacologiques agissant entre autres au niveau cellulaire. Recemment, la detection basee sur la resonance des plasmons de surface (SPR) a ete appliquee a l'etude de reponses cellulaires. Cette methode de detection, permettant d'observer des variations d'indice de refraction associes a de faibles changements de masse a la surface d'un metal, a l'avantage de permettre l'etude d'une population de cellules vivantes en temps reel, sans necessiter l'introduction d'agents de marquage. Pour effectuer la detection au niveau de cellules individuelles, on peut employer la microscopie SPR, qui consiste a localiser spatialement la detection par un systeme d'imagerie. Cependant, la detection basee sur la SPR est une mesure sans marquage et les signaux mesures sont attribues a une reponse moyennee des differentes sources cellulaires. Afin de mieux comprendre et identifier les composantes cellulaires generant le signal mesure en SPR, il est pertinent de combiner la microscopie SPR avec une modalite complementaire, soit l'imagerie de fluorescence. C'est dans cette problematique que s'insere ce projet de these, consistant a concevoir deux plates-formes distinctes de microscopie SPR et de fluorescence optimisees pour l'etude cellulaire, de sorte a evaluer les possibilites d'integration de ces deux modalites en un seul systeme. Des substrats adaptes pour chaque plate-forme ont ete concus et realises. Ces substrats employaient une couche d'argent passivee par l'ajout d'une mince couche d'or. La stabilite et la biocompatibilite des substrats ont ete validees pour l'etude cellulaire. Deux configurations permettant d'ameliorer la sensibilite en sondant les cellules plus profondement ont ete evaluees, soit l'emploi de plasmons de surface a longue portee et de guides d'onde a gaine metallique. La sensibilite accrue de ces configurations a aussi ete demontree pour un usage en biodetection cellulaire. Une plate-forme permettant de mesurer la spectroscopie SPR simultanement avec l'acquisition d'images de fluorescence a ete realisee. Cette plate-forme a ensuite ete validee par l'etude de reponses cellulaires suite a une stimulation pharmacologique. Puis, un systeme base sur la microscopie SPR a ete concu et caracterise. Son emploi pour l'etude de reponses au niveau de cellules individuelles a ete demontre. Finalement, les forces et faiblesses des substrats et des plates-formes realisees au cours de la these ont ete evaluees. Des possibilites d'amelioration sont mises de l'avant et l'integration des modalites de microscopie SPR et de fluorescence suite aux travaux de la these est discutee. Les realisations au cours de cette etude ont donc permis d'identifier les composantes cellulaires impliquees dans la generation du signal mesure en biodetection SPR. Mots-cles : Resonance des plasmons de surface, microscopie SPR, plasmons de surface a longue portee LRSPR, guide d'onde a gaine metallique MCWG, fluorescence exaltee par plasmons de surface SPEF, biodetection cellulaire, imagerie SPR.

Video-rate resonant scanning multiphoton microscopy

PubMed Central

Kirkpatrick, Nathaniel D.; Chung, Euiheon; Cook, Daniel C.; Han, Xiaoxing; Gruionu, Gabriel; Liao, Shan; Munn, Lance L.; Padera, Timothy P.; Fukumura, Dai; Jain, Rakesh K.

2013-01-01

The abnormal tumor microenvironment fuels tumor progression, metastasis, immune suppression, and treatment resistance. Over last several decades, developments in and applications of intravital microscopy have provided unprecedented insights into the dynamics of the tumor microenvironment. In particular, intravital multiphoton microscopy has revealed the abnormal structure and function of tumor-associated blood and lymphatic vessels, the role of aberrant tumor matrix in drug delivery, invasion and metastasis of tumor cells, the dynamics of immune cell trafficking to and within tumors, and gene expression in tumors. However, traditional multiphoton microscopy suffers from inherently slow imaging rates—only a few frames per second, thus unable to capture more rapid events such as blood flow, lymphatic flow, and cell movement within vessels. Here, we report the development and implementation of a video-rate multiphoton microscope (VR-MPLSM) based on resonant galvanometer mirror scanning that is capable of recording at 30 frames per second and acquiring intravital multispectral images. We show that the design of the system can be readily implemented and is adaptable to various experimental models. As examples, we demonstrate the utility of the system to directly measure flow within tumors, capture metastatic cancer cells moving within the brain vasculature and cells in lymphatic vessels, and image acute responses to changes in a vascular network. VR-MPLSM thus has the potential to further advance intravital imaging and provide new insight into the biology of the tumor microenvironment. PMID:24353926

Learning from connectomics on the fly.

PubMed

Schlegel, Philipp; Costa, Marta; Jefferis, Gregory Sxe

2017-12-01

Parallels between invertebrates and vertebrates in nervous system development, organisation and circuits are powerful reasons to use insects to study the mechanistic basis of behaviour. The last few years have seen the generation in Drosophila melanogaster of very large light microscopy data sets, genetic driver lines and tools to report or manipulate neural activity. These resources in conjunction with computational tools are enabling large scale characterisation of neuronal types and their functional properties. These are complemented by 3D electron microscopy, providing synaptic resolution data. A whole brain connectome of the fly larva is approaching completion based on manual reconstruction of electron-microscopy data. An adult whole brain dataset is already publicly available and focussed reconstruction is under way, but its 40× greater volume would require ∼500-5000 person-years of manual labour. Nevertheless rapid technical improvements in imaging and especially automated segmentation will likely deliver a complete adult connectome in the next 5 years. To enhance our understanding of the circuit basis of behaviour, light and electron microscopy outputs must be integrated with functional and physiological information into comprehensive databases. We review presently available data, tools and opportunities in Drosophila. We then consider the limits and potential of future progress and how this may impact neuroscience in rich model systems provided by larger insects and vertebrates. Copyright © 2017 Elsevier Inc. All rights reserved.

Metabolic Mapping of Breast Cancer with Multiphoton Spectral and Lifetime Imaging

DTIC Science & Technology

2007-03-01

spectral and lifetime characterization of NADH may be used to reveal metabolic changes in vivo and has potential to be used as an early diagnostic...combined spectral lifetime imaging modality will help for 5 characterization of breast cancer cells from cell culture based models to a relevant in... spectral and lifetime system and integrated into a multiphoton fluorescence excitation microscopy system 7 • Calibrated and characterized this

Microemulsions containing lecithin and sugar-based surfactants: nanoparticle templates for delivery of proteins and peptides.

PubMed

Graf, Anja; Ablinger, Elisabeth; Peters, Silvia; Zimmer, Andreas; Hook, Sarah; Rades, Thomas

2008-02-28

Two pseudo-ternary systems comprising isopropyl myristate, soybean lecithin, water, ethanol and either decyl glucoside (DG) or capryl-caprylyl glucoside (CCG) as surfactant were investigated for their potential to form microemulsion templates to produce nanoparticles as drug delivery vehicles for proteins and peptides. All microemulsion and nanoparticle compounds used were pharmaceutically acceptable and biocompatible. Phase diagrams were established and characterized using polarizing light microscopy, viscosity, conductivity, electron microscopy, differential scanning calorimetry and self-diffusion NMR. An area in the phase diagrams containing optically isotropic, monophasic systems was designated as the microemulsion region and systems therein identified as solution-type microemulsions. Poly(alkylcyanoacrylate) nanoparticles prepared by interfacial polymerisation from selected microemulsions ranged from 145 to 660nm in size with a unimodal size distribution depending on the type of monomer (ethyl (2) or butyl (2) cyanoacrylate) and microemulsion template. Generally larger nanoparticles were formed by butyl (2) cyanoacrylate. Insulin was added as a model protein and did not alter the physicochemical behaviour of the microemulsions or the morphology of the nanoparticles. However, insulin-loaded nanoparticles in the CCG containing system decreased in size when using butyl (2) cyanoacrylate. This study shows that microemulsions containing sugar-based surfactants are suitable formulation templates for the formation of nanoparticles to deliver peptides.

Superresolution microscopy for microbiology

PubMed Central

Coltharp, Carla; Xiao, Jie

2014-01-01

Summary This review provides a practical introduction to superresolution microscopy from the perspective of microbiological research. Because of the small sizes of bacterial cells, superresolution methods are particularly powerful and suitable for revealing details of cellular structures that are not resolvable under conventional fluorescence light microscopy. Here we describe the methodological concepts behind three major categories of super-resolution light microscopy: photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) and stimulated emission-depletion (STED) microscopy. We then present recent applications of each of these techniques to microbial systems, which have revealed novel conformations of cellular structures and described new properties of in vivo protein function and interactions. Finally, we discuss the unique issues related to implementing each of these superresolution techniques with bacterial specimens and suggest avenues for future development. The goal of this review is to provide the necessary technical background for interested microbiologists to choose the appropriate super-resolution method for their biological systems, and to introduce the practical considerations required for designing and analysing superresolution imaging experiments. PMID:22947061

Rapid analysis and exploration of fluorescence microscopy images.

PubMed

Pavie, Benjamin; Rajaram, Satwik; Ouyang, Austin; Altschuler, Jason M; Steininger, Robert J; Wu, Lani F; Altschuler, Steven J

2014-03-19

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard. Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Two-component gelator isomers with different combination of amine and acid: Helical/non-helical morphology and selective adsorption of dyes.

PubMed

Han, Xiaoyu; Liu, Jiahui; Zhao, Chaoyue; Zhang, Bao; Xu, Xiufang; Song, Jian

2018-09-01

Hydrogels induced by two-component gelator isomers based on the different amine/acid interactions were investigated. Scanning electron microscopy and atomic force microscopy images of the xerogel obtained from the two hydrogels revealed different assembly morphologies. While left-handed helical fibers were observed for the amine-acid based xerogel, acid-amine underwent self-assembly to afford smooth fibers. Fourier transform infrared spectroscopy, fluorescence, and X-ray diffraction measurements combined with density functional theory calculations suggested that the different self-assembly patterns of gelators resulted in opposite electric charges on the xerogel surfaces, in line with Zeta potential measurements. Based on these opposite charges resulting from their different self-assemblies, both xerogels demonstrated efficient dye adsorption abilities with different selectivities. Interestingly, the adsorption performance was not influenced by the salt in the dye solution. Furthermore, the xerogels still showed high dye adsorption efficiency after four cycles. These results provide a two-component hydrogel method for the purification of dye-polluted water systems, while also paving the way for future design of functionalized supramolecular self-assembly systems. Copyright © 2018 Elsevier Inc. All rights reserved.

Photo-induced processes in collagen-hypericin system revealed by fluorescence spectroscopy and multiphoton microscopy

PubMed Central

Hovhannisyan, V.; Guo, H. W.; Hovhannisyan, A.; Ghukasyan, V.; Buryakina, T.; Chen, Y. F.; Dong, C. Y.

2014-01-01

Collagen is the main structural protein and the key determinant of mechanical and functional properties of tissues and organs. Proper balance between synthesis and degradation of collagen molecules is critical for maintaining normal physiological functions. In addition, collagen influences tumor development and drug delivery, which makes it a potential cancer therapy target. Using second harmonic generation, two-photon excited fluorescence microscopy, and spectrofluorimetry, we show that the natural pigment hypericin induces photosensitized destruction of collagen-based tissues. We demonstrate that hypericin–mediated processes in collagen fibers are irreversible and may be used for the treatment of cancer and collagen-related disorders. PMID:24877000

Photo-induced processes in collagen-hypericin system revealed by fluorescence spectroscopy and multiphoton microscopy.

PubMed

Hovhannisyan, V; Guo, H W; Hovhannisyan, A; Ghukasyan, V; Buryakina, T; Chen, Y F; Dong, C Y

2014-05-01

Collagen is the main structural protein and the key determinant of mechanical and functional properties of tissues and organs. Proper balance between synthesis and degradation of collagen molecules is critical for maintaining normal physiological functions. In addition, collagen influences tumor development and drug delivery, which makes it a potential cancer therapy target. Using second harmonic generation, two-photon excited fluorescence microscopy, and spectrofluorimetry, we show that the natural pigment hypericin induces photosensitized destruction of collagen-based tissues. We demonstrate that hypericin-mediated processes in collagen fibers are irreversible and may be used for the treatment of cancer and collagen-related disorders.

Invited review article: high-speed flexure-guided nanopositioning: mechanical design and control issues.

PubMed

Yong, Y K; Moheimani, S O R; Kenton, B J; Leang, K K

2012-12-01

Recent interest in high-speed scanning probe microscopy for high-throughput applications including video-rate atomic force microscopy and probe-based nanofabrication has sparked attention on the development of high-bandwidth flexure-guided nanopositioning systems (nanopositioners). Such nanopositioners are designed to move samples with sub-nanometer resolution with positioning bandwidth in the kilohertz range. State-of-the-art designs incorporate uniquely designed flexure mechanisms driven by compact and stiff piezoelectric actuators. This paper surveys key advances in mechanical design and control of dynamic effects and nonlinearities, in the context of high-speed nanopositioning. Future challenges and research topics are also discussed.

A flexible and rapid frequency selective scheme for SRS microscopy

NASA Astrophysics Data System (ADS)

Li, Jingting; Yue, Yuankai; Shih, Wei-Chuan

2017-02-01

Stimulated Raman scattering (SRS) is a label-free imaging technique suitable for studying biological systems. Due to stimulated nature by ultrafast laser pulses, SRS microscopy has the advantage of significantly higher sensitivity but often reduced spectroscopic information. In this paper, we present a newly constructed femtosecond SRS microscope with a high-speed dynamic micromirror device based pulse shaper to achieve flexible and rapid frequency selection within the C-H stretch region near 2800 to 3100 cm-1 with spectral width of 30 cm-1. This technique is applicable to lipid profiling such as cell activity mapping, lipid distribution mapping and distinction among subclasses.

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

Facility for low-temperature spin-polarized-scanning tunneling microscopy studies of magnetic/spintronic materials prepared in situ by nitride molecular beam epitaxy.

PubMed

Lin, Wenzhi; Foley, Andrew; Alam, Khan; Wang, Kangkang; Liu, Yinghao; Chen, Tianjiao; Pak, Jeongihm; Smith, Arthur R

2014-04-01

Based on the interest in, as well as exciting outlook for, nitride semiconductor based structures with regard to electronic, optoelectronic, and spintronic applications, it is compelling to investigate these systems using the powerful technique of spin-polarized scanning tunneling microscopy (STM), a technique capable of achieving magnetic resolution down to the atomic scale. However, the delicate surfaces of these materials are easily corrupted by in-air transfers, making it unfeasible to study them in stand-alone ultra-high vacuum STM facilities. Therefore, we have carried out the development of a hybrid system including a nitrogen plasma assisted molecular beam epitaxy/pulsed laser epitaxy facility for sample growth combined with a low-temperature, spin-polarized scanning tunneling microscope system. The custom-designed molecular beam epitaxy growth system supports up to eight sources, including up to seven effusion cells plus a radio frequency nitrogen plasma source, for epitaxially growing a variety of materials, such as nitride semiconductors, magnetic materials, and their hetero-structures, and also incorporating in situ reflection high energy electron diffraction. The growth system also enables integration of pulsed laser epitaxy. The STM unit has a modular design, consisting of an upper body and a lower body. The upper body contains the coarse approach mechanism and the scanner unit, while the lower body accepts molecular beam epitaxy grown samples using compression springs and sample skis. The design of the system employs two stages of vibration isolation as well as a layer of acoustic noise isolation in order to reduce noise during STM measurements. This isolation allows the system to effectively acquire STM data in a typical lab space, which during its construction had no special and highly costly elements included, (such as isolated slabs) which would lower the environmental noise. The design further enables tip exchange and tip coating without breaking vacuum, and convenient visual access to the sample and tip inside a superconducting magnet cryostat. A sample/tip handling system is optimized for both the molecular beam epitaxy growth system and the scanning tunneling microscope system. The sample/tip handing system enables in situ STM studies on epitaxially grown samples, and tip exchange in the superconducting magnet cryostat. The hybrid molecular beam epitaxy and low temperature scanning tunneling microscopy system is capable of growing semiconductor-based hetero-structures with controlled accuracy down to a single atomic-layer and imaging them down to atomic resolution.

Automated analysis of time-lapse fluorescence microscopy images: from live cell images to intracellular foci.

PubMed

Dzyubachyk, Oleh; Essers, Jeroen; van Cappellen, Wiggert A; Baldeyron, Céline; Inagaki, Akiko; Niessen, Wiro J; Meijering, Erik

2010-10-01

Complete, accurate and reproducible analysis of intracellular foci from fluorescence microscopy image sequences of live cells requires full automation of all processing steps involved: cell segmentation and tracking followed by foci segmentation and pattern analysis. Integrated systems for this purpose are lacking. Extending our previous work in cell segmentation and tracking, we developed a new system for performing fully automated analysis of fluorescent foci in single cells. The system was validated by applying it to two common tasks: intracellular foci counting (in DNA damage repair experiments) and cell-phase identification based on foci pattern analysis (in DNA replication experiments). Experimental results show that the system performs comparably to expert human observers. Thus, it may replace tedious manual analyses for the considered tasks, and enables high-content screening. The described system was implemented in MATLAB (The MathWorks, Inc., USA) and compiled to run within the MATLAB environment. The routines together with four sample datasets are available at http://celmia.bigr.nl/. The software is planned for public release, free of charge for non-commercial use, after publication of this article.

Miniature fiber-optic multiphoton microscopy system using frequency-doubled femtosecond Er-doped fiber laser

PubMed Central

Huang, Lin; Mills, Arthur K.; Zhao, Yuan; Jones, David J.; Tang, Shuo

2016-01-01

We report on a miniature fiber-optic multiphoton microscopy (MPM) system based on a frequency-doubled femtosecond Er-doped fiber laser. The femtosecond pulses from the laser source are delivered to the miniature fiber-optic probe at 1.58 µm wavelength, where a standard single mode fiber is used for delivery without the need of free-space dispersion compensation components. The beam is frequency-doubled inside the probe by a periodically poled MgO:LiNbO3 crystal. Frequency-doubled pulses at 786 nm with a maximum power of 80 mW and a pulsewidth of 150 fs are obtained and applied to excite intrinsic signals from tissues. A MEMS scanner, a miniature objective, and a multimode collection fiber are further used to make the probe compact. The miniature fiber-optic MPM system is highly portable and robust. Ex vivo multiphoton imaging of mammalian skins demonstrates the capability of the system in imaging biological tissues. The results show that the miniature fiber-optic MPM system using frequency-doubled femtosecond fiber laser can potentially bring the MPM imaging for clinical applications. PMID:27231633

Two-Photon Fluorescence Microscope for Microgravity Research

NASA Technical Reports Server (NTRS)

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

2005-01-01

A two-photon fluorescence microscope has been developed for the study of biophysical phenomena. Two-photon microscopy is a novel form of laser-based scanning microscopy that enables three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon optical microscopy, two-photon microscopy utilizes the simultaneous nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption, so an ultra-fast pulsed laser source is typically employed. On the other hand, the critical energy threshold for two-photon absorption leads to fluorophore excitation that is intrinsically localized to the focal volume. Consequently, two-photon microscopy enables optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction (relative to one-photon optical microscopy) in photon-induced damage because of the longer excitation wavelength. This reduction is especially advantageous for in vivo studies. Relative to confocal microscopy, there is also a reduction in background fluorescence, and, because of a reduction in Rayleigh scattering, there is a 4 increase of penetration depth. The prohibitive cost of a commercial two-photon fluorescence-microscope system, as well as a need for modularity, has led to the construction of a custom-built system (see Figure 1). This system includes a coherent mode-locked titanium: sapphire laser emitting 120-fs-duration pulses at a repetition rate of 80 MHz. The pulsed laser has an average output power of 800 mW and a wavelength tuning range of 700 to 980 nm, enabling the excitation of a variety of targeted fluorophores. The output from the laser is attenuated, spatially filtered, and then directed into a confocal scanning head that has been modified to provide for side entry of the laser beam. The laser output coupler has been replaced with a dichroic filter that reflects the longer-wavelength excitation light and passes the shorter-wavelength fluorescence light. Also, the confocal pinhole has been removed to increase the signal strength. The laser beam is scanned by a twoperpendicular- axis pair of galvanometer mirrors through a pupil transfer lens into the side port of an inverted microscope. Finally, the beam is focused by a 63-magnification, 1.3-numerical- aperture oil-immersion objective lens onto a specimen. The pupil transfer lens serves to match the intermediate image planes of the scanning head and the microscope, and its location is critical. In order to maximize the quality of the image, (that is, the point spread function of the objective lens for all scan positions), the entire system was modeled in optical-design software, and the various free design parameters (the parameters of the spatial-filter components as well as the separations of all of the system components) were determined through an iterative optimization process. A modular design was chosen to facilitate access to the optical train for future fluorescence correlation spectroscopy and fluorescence-lifetime experiments.

Characterisation of phases in nanostructured, multilayered titanium alloys by analytical and high-resolution electron microscopy.

PubMed

Czyrska-Filemonowicz, A; Buffat, P A

2009-01-01

Surface processing of a Ti-6Al-4V alloy led to a complex multilayered microstructure containing several phases of the Ni-Ti-P-Al-O system, which improves the mechanical and tribological surface properties. The microstructure, chemical and phase compositions of the hard layer formed on the surface were investigated by LM, XRD, SEM as well as analytical/high-resolution TEM, STEM, EDS, electron diffraction and FIB. Phase identification based on electron diffraction, HRTEM and EDS microanalysis revealed the presence of several binary and ternary phases in the system Ti-Ni-P, sometimes with partial substitution of Ti by Al. However some phases, mainly nanoparticles, still remain not identified satisfactorily. Electron microscopy techniques used for identification of phases present in surface multilayers and some practical limits to their routine application are reminded here.

Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time.

PubMed

Kong, Muwen; Beckwitt, Emily C; Springall, Luke; Kad, Neil M; Van Houten, Bennett

2017-01-01

Single-molecule approaches to solving biophysical problems are powerful tools that allow static and dynamic real-time observations of specific molecular interactions of interest in the absence of ensemble-averaging effects. Here, we provide detailed protocols for building an experimental system that employs atomic force microscopy and a single-molecule DNA tightrope assay based on oblique angle illumination fluorescence microscopy. Together with approaches for engineering site-specific lesions into DNA substrates, these complementary biophysical techniques are well suited for investigating protein-DNA interactions that involve target-specific DNA-binding proteins, such as those engaged in a variety of DNA repair pathways. In this chapter, we demonstrate the utility of the platform by applying these techniques in the studies of proteins participating in nucleotide excision repair. © 2017 Elsevier Inc. All rights reserved.

Phase sensitive optical coherence microscopy for photothermal imaging of gold nanorods

NASA Astrophysics Data System (ADS)

Hu, Yong; Podoleanu, Adrian G.; Dobre, George

2018-03-01

We describe a swept source based phase sensitive optical coherence microscopy (OCM) system for photothermal imaging of gold nanorods (GNR). The phase sensitive OCM system employed in the study has a displacement sensitivity of 0.17 nm to vibrations at single frequencies below 250 Hz. We demonstrate the generation of phase maps and confocal phase images. By displaying the difference between successive confocal phase images, we perform the confocal photothermal imaging of accumulated GNRs behind a glass coverslip and behind the scattering media separately. Compared with two-photon luminescence (TPL) detection techniques reported in literature, the technique in this study has the advantage of a simplified experimental setup and provides a more efficient method for imaging the aggregation of GNR. However, the repeatability performance of this technique suffers due to jitter noise from the swept laser source.

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

PubMed Central

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

2016-01-01

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

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

PubMed Central

Etheridge, Thomas J.; Boulineau, Rémi L.; Herbert, Alex; Watson, Adam T.; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest; Osborne, Mark A.; Klenerman, David; Lee, Steven F.; Carr, Antony M.

2014-01-01

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds. PMID:25106872

Combining atomic force and fluorescence microscopy for analysis of quantum-dot labeled protein–DNA complexes

PubMed Central

Ebenstein, Yuval; Gassman, Natalie; Kim, Soohong; Weiss, Shimon

2011-01-01

Atomic force microscopy (AFM) and fluorescence microscopy are widely used for the study of protein-DNA interactions. While AFM excels in its ability to elucidate structural detail and spatial arrangement, it lacks the ability to distinguish between similarly sized objects in a complex system. This information is readily accessible to optical imaging techniques via site-specific fluorescent labels, which enable the direct detection and identification of multiple components simultaneously. Here, we show how the utilization of semiconductor quantum dots (QDs), serving as contrast agents for both AFM topography and fluorescence imaging, facilitates the combination of both imaging techniques, and with the addition of a flow based DNA extension method for sample deposition, results in a powerful tool for the study of protein-DNA complexes. We demonstrate the inherent advantages of this novel combination of techniques by imaging individual RNA polymerases (RNAP) on T7 genomic DNA. PMID:19452448

Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy.

PubMed

Quan, Xiangyu; Matoba, Osamu; Awatsuji, Yasuhiro

2017-05-01

A technique of three-dimensional (3D) intensity retrieval from defocused, two-dimensional (2D) fluorescent images in the multimodal digital holographic microscopy (DHM) is proposed. In the multimodal DHM, 3D phase and 2D fluorescence distributions are obtained simultaneously by an integrated system of an off-axis DHM and a conventional epifluorescence microscopy, respectively. This gives us more information of the target; however, defocused fluorescent images are observed due to the short depth of field. In this Letter, we propose a method to recover the defocused images based on the phase compensation and backpropagation from the defocused plane to the focused plane using the distance information that is obtained from a 3D phase distribution. By applying Zernike polynomial phase correction, we brought back the fluorescence intensity to the focused imaging planes. The experimental demonstration using fluorescent beads is presented, and the expected applications are suggested.

Automated aberration compensation in high numerical aperture systems for arbitrary laser modes (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hering, Julian; Waller, Erik H.; von Freymann, Georg

2017-02-01

Since a large number of optical systems and devices are based on differently shaped focal intensity distributions (point-spread-functions, PSF), the PSF's quality is crucial for the application's performance. E.g., optical tweezers, optical potentials for trapping of ultracold atoms as well as stimulated-emission-depletion (STED) based microscopy and lithography rely on precisely controlled intensity distributions. However, especially in high numerical aperture (NA) systems, such complex laser modes are easily distorted by aberrations leading to performance losses. Although different approaches addressing phase retrieval algorithms have been recently presented[1-3], fast and automated aberration compensation for a broad variety of complex shaped PSFs in high NA systems is still missing. Here, we report on a Gerchberg-Saxton[4] based algorithm (GSA) for automated aberration correction of arbitrary PSFs, especially for high NA systems. Deviations between the desired target intensity distribution and the three-dimensionally (3D) scanned experimental focal intensity distribution are used to calculate a correction phase pattern. The target phase distribution plus the correction pattern are displayed on a phase-only spatial-light-modulator (SLM). Focused by a high NA objective, experimental 3D scans of several intensity distributions allow for characterization of the algorithms performance: aberrations are reliably identified and compensated within less than 10 iterations. References 1. B. M. Hanser, M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Phase-retrieved pupil functions in wide-field fluorescence microscopy," J. of Microscopy 216(1), 32-48 (2004). 2. A. Jesacher, A. Schwaighofer, S. Frhapter, C. Maurer, S. Bernet, and M. Ritsch-Marte, "Wavefront correction of spatial light modulators using an optical vortex image," Opt. Express 15(9), 5801-5808 (2007). 3. A. Jesacher and M. J. Booth, "Parallel direct laser writing in three dimensions with spatially dependent aberration correction," Opt. Express 18(20), 21090-21099 (2010). 4. R. W. Gerchberg and W. O. Saxton, "A practical algorithm for the determination of the phase from image and diffraction plane pictures," Optik 35(2), 237-246 (1972).

Adaptive optics in multiphoton microscopy: comparison of two, three and four photon fluorescence

PubMed Central

Sinefeld, David; Paudel, Hari P.; Ouzounov, Dimitre G.; Bifano, Thomas G.; Xu, Chris

2015-01-01

We demonstrate adaptive optics system based on nonlinear feedback from 3- and 4-photon fluorescence. The system is based on femtosecond pulses created by soliton self-frequency shift of a 1550-nm fiber-based femtosecond laser together with micro-electro-mechanical system (MEMS) phase spatial light modulator (SLM). We perturb the 1020-segment SLM using an orthogonal Walsh sequence basis set with a modified version of three-point phase shifting interferometry. We show the improvement after aberrations correction in 3-photon signal from fluorescent beads. In addition, we compare the improvement obtained in the same adaptive optical system for 2-, 3- and 4-photon fluorescence using dye pool. We show that signal improvement resulting from aberration correction grows exponentially as a function of the order of nonlinearity. PMID:26698772

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

PubMed

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

2004-07-01

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

Low-cost mobile phone microscopy with a reversed mobile phone camera lens.

PubMed

Switz, Neil A; D'Ambrosio, Michael V; Fletcher, Daniel A

2014-01-01

The increasing capabilities and ubiquity of mobile phones and their associated digital cameras offer the possibility of extending low-cost, portable diagnostic microscopy to underserved and low-resource areas. However, mobile phone microscopes created by adding magnifying optics to the phone's camera module have been unable to make use of the full image sensor due to the specialized design of the embedded camera lens, exacerbating the tradeoff between resolution and field of view inherent to optical systems. This tradeoff is acutely felt for diagnostic applications, where the speed and cost of image-based diagnosis is related to the area of the sample that can be viewed at sufficient resolution. Here we present a simple and low-cost approach to mobile phone microscopy that uses a reversed mobile phone camera lens added to an intact mobile phone to enable high quality imaging over a significantly larger field of view than standard microscopy. We demonstrate use of the reversed lens mobile phone microscope to identify red and white blood cells in blood smears and soil-transmitted helminth eggs in stool samples.

Confocal microscopy refines generic concept of a problematic taxon: rediagnosis of the genus Neoprothrix and remarks on female anatomy of eriophyoids (Acari: Eriophyoidea).

PubMed

Chetverikov, Philipp E; Desnitskiy, Alexey G; Navia, Denise

2015-02-16

Due to the higher resolution, confocal microscopy (CLSM) can be applied to refine the origin of tiny structures of the autofluorescent exoskeletons of microarthropods (mites in particular) which are hard to visualize using traditional differential interference contract light microscopy (DIC LM) and phase contrast light microscopy (PC LM). Three-dimensional (3D) reconstructions of the prodorsal shield topography of eriophyoid mites using Neoprothrix hibiscus Reis and Navia as a model, suggest that the structures originally treated as paired setae vi are two internal rod-like apodemes. Based on this, the genus Neoprothrix is excluded from the subfamily Prothricinae Amrine and transferred to the subfamily Sierraphytoptinae Keifer. Observations on partially cleared specimens of N. hibiscus showed that remnants of the central nervous system, paired glands and developing oocytes can be visualized using DIC LM and CLSM methods. New high quality microscope images are provided of recently described "flower-shaped" structures and two main components of yolk inclusions of the mature eggs inside the oviduct.

Low-Cost Mobile Phone Microscopy with a Reversed Mobile Phone Camera Lens

PubMed Central

Fletcher, Daniel A.

2014-01-01

The increasing capabilities and ubiquity of mobile phones and their associated digital cameras offer the possibility of extending low-cost, portable diagnostic microscopy to underserved and low-resource areas. However, mobile phone microscopes created by adding magnifying optics to the phone's camera module have been unable to make use of the full image sensor due to the specialized design of the embedded camera lens, exacerbating the tradeoff between resolution and field of view inherent to optical systems. This tradeoff is acutely felt for diagnostic applications, where the speed and cost of image-based diagnosis is related to the area of the sample that can be viewed at sufficient resolution. Here we present a simple and low-cost approach to mobile phone microscopy that uses a reversed mobile phone camera lens added to an intact mobile phone to enable high quality imaging over a significantly larger field of view than standard microscopy. We demonstrate use of the reversed lens mobile phone microscope to identify red and white blood cells in blood smears and soil-transmitted helminth eggs in stool samples. PMID:24854188

Single molecule microscopy in 3D cell cultures and tissues.

PubMed

Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

2014-12-15

From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

A comparative laboratory diagnosis of malaria: microscopy versus rapid diagnostic test kits.

PubMed

Azikiwe, C C A; Ifezulike, C C; Siminialayi, I M; Amazu, L U; Enye, J C; Nwakwunite, O E

2012-04-01

To compare the two methods of rapid diagnostic tests (RDTs) and microscopy in the diagnosis of malaria. RDTs and microscopy were carried out to diagnose malaria. Percentage malaria parasitaemia was calculated on thin films and all non-acute cases of plasmodiasis with less than 0.001% malaria parasitaemia were regarded as negative. Results were simply presented as percentage positive of the total number of patients under study. The results of RDTs were compared to those of microscopy while those of RDTs based on antigen were compared to those of RDTs based on antibody. Patients' follow-up was made for all cases. All the 200 patients under present study tested positive to RDTs based on malaria antibodies (serum) method (100%). 128 out of 200 tested positive to RDTs based on malaria antigen (whole blood) method (64%), while 118 out of 200 patients under present study tested positive to visual microscopy of Lieshman and diluted Giemsa (59%). All patients that tested positive to microscopy also tested positive to RDTs based on antigen. All patients on the second day of follow-up were non-febrile and had antimalaria drugs. We conclude based on the present study that the RDTs based on malaria antigen (whole blood) method is as specific as the traditional microscopy and even appears more sensitive than microscopy. The RDTs based on antibody (serum) method is unspecific thus it should not be encouraged. It is most likely that Africa being an endemic region, formation of certain levels of malaria antibody may not be uncommon. The present study also supports the opinion that a good number of febrile cases is not due to malaria. We support WHO's report on cost effectiveness of RDTs but, recommend that only the antigen based method should possibly, be adopted in Africa and other malaria endemic regions of the world.

Fourier Ptychographic Microscopy for Rapid, High-Resolution Imaging of Circulating Tumor Cells Enriched by Microfiltration.

PubMed

Williams, Anthony; Chung, Jaebum; Yang, Changhuei; Cote, Richard J

2017-01-01

Examining the hematogenous compartment for evidence of metastasis has increased significantly within the oncology research community in recent years, due to the development of technologies aimed at the enrichment of circulating tumor cells (CTCs), the subpopulation of primary tumor cells that gain access to the circulatory system and are responsible for colonization at distant sites. In contrast to other technologies, filtration-based CTC enrichment, which exploits differences in size between larger tumor cells and surrounding smaller, non-tumor blood cells, has the potential to improve CTC characterization through isolation of tumor cell populations with greater molecular heterogeneity. However, microscopic analysis of uneven filtration surfaces containing CTCs is laborious, time-consuming, and inconsistent, preventing widespread use of filtration-based enrichment technologies. Here, integrated with a microfiltration-based CTC and rare cell enrichment device we have previously described, we present a protocol for Fourier Ptychographic Microscopy (FPM), a method that, unlike many automated imaging platforms, produces high-speed, high-resolution images that can be digitally refocused, allowing users to observe objects of interest present on multiple focal planes within the same image frame. The development of a cost-effective and high-throughput CTC analysis system for filtration-based enrichment technologies could have profound clinical implications for improved CTC detection and analysis.

Super-resolution photoacoustic microscopy using a localized near-field of a plasmonic nanoaperture: a three-dimensional simulation study

NASA Astrophysics Data System (ADS)

Park, Byullee; Lee, Hongki; Upputuri, Paul Kumar; Pramanik, Manojit; Kim, Donghyun; Kim, Chulhong

2018-02-01

Super-resolution microscopy has been increasingly important to delineate nanoscale biological structures or nanoparticles. With these increasing demands, several imaging modalities, including super-resolution fluorescence microscope (SRFM) and electron microscope (EM), have been developed and commercialized. These modalities achieve nanoscale resolution, however, SRFM cannot image without fluorescence, and sample preparation of EM is not suitable for biological specimens. To overcome those disadvantages, we have numerically studied the possibility of superresolution photoacoustic microscopy (SR-PAM) based on near-field localization of light. Photoacoustic (PA) signal is generally acquired based on optical absorption contrast; thus it requires no agents or pre-processing for the samples. The lateral resolution of the conventional photoacoustic microscopy is limited to 200 nm by diffraction limit, therefore reducing the lateral resolution is a major research impetus. Our approach to breaking resolution limit is to use laser pulses of extremely small spot size as a light source. In this research, we simulated the PA signal by constructing the three dimensional SR-PAM system environment using the k-Wave toolbox. As the light source, we simulated ultrashort light pulses using geometrical nanoaperture with near-field localization of surface plasmons. Through the PA simulation, we have successfully distinguish cuboids spaced 3 nm apart. In the near future, we will develop the SR-PAM and it will contribute to biomedical and material sciences.

Plasmonics Enhanced Smartphone Fluorescence Microscopy.

PubMed

Wei, Qingshan; Acuna, Guillermo; Kim, Seungkyeum; Vietz, Carolin; Tseng, Derek; Chae, Jongjae; Shir, Daniel; Luo, Wei; Tinnefeld, Philip; Ozcan, Aydogan

2017-05-18

Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

Computational method for multi-modal microscopy based on transport of intensity equation

NASA Astrophysics Data System (ADS)

Li, Jiaji; Chen, Qian; Sun, Jiasong; Zhang, Jialin; Zuo, Chao

2017-02-01

In this paper, we develop the requisite theory to describe a hybrid virtual-physical multi-modal imaging system which yields quantitative phase, Zernike phase contrast, differential interference contrast (DIC), and light field moment imaging simultaneously based on transport of intensity equation(TIE). We then give the experimental demonstration of these ideas by time-lapse imaging of live HeLa cell mitosis. Experimental results verify that a tunable lens based TIE system, combined with the appropriate post-processing algorithm, can achieve a variety of promising imaging modalities in parallel with the quantitative phase images for the dynamic study of cellular processes.

Microlenses and microcameras for biomedical imaging

NASA Astrophysics Data System (ADS)

Kanhere, Aditi

Liquid lens technology is a rapidly progressing field driven by the promise of low cost fabrication, faster response, fewer mechanical elements, versatility and ease of customization for different applications. Here we present the use of liquid lenses for biomedical optics and medical imaging. I will specifically focus on our approaches towards the development of two liquid-lens optical systems -- laparoscopic cameras and 3D microscopy. The first part of this work is based on the development of a multi-camera laparoscopic imaging system with tunable focusing capability. The work attempts to find a solution to overcome many of the fundamental challenges faced by current laparoscopic imaging systems. The system is developed upon the key idea that widely spread multiple, tunable microcameras can cover a large range of vantage points and field of view (FoV) for intra-abdominal visualization. Our design features multiple tunable-focus microcameras integrated with a surgical port to provide panoramic intra-abdominal visualization with enhanced depth perception. Our system can be optically tuned to focus in on objects within a range of 5 mm to infinity, with a FoV adjustable between 36 degrees and 130 degrees. This unique approach also eliminates the requirement of an exclusive imaging port and need for navigation of cameras between ports during surgery. The second part of this report focuses on the application of tunable lenses in microscopy. Conventional wide-field microscopy is one of the most widely used optical microscopy technique. This technique typically captures a two dimensional image of a specimen. For a volumetric visualization of the sample or to enable depth scanning along the axial direction, it is necessary to move the sample relative to the fixed focal plane of the microscope objective. For this purpose, a mechanical z-scanning stage is typically employed. The stage enables the focal plane to move through the sample. Typical approaches used to achieve axial scanning are a motorized stepper stage or a piezoelectric stage. While stepper motors offer the advantage of unlimited travel distance, they suffer from hysteresis. Piezoelectric stages on the other hand, help eliminate hysteresis at the cost of the travel distance which is reduced to 100-200 mum. Both the types of stages, however, are bulky and cause vibrations and wobble in the sample due to high inertia. Additional care is required to avoid mechanical overshoots and backlash from the tip touching the sample. Additionally, for water or oil-immersion lenses, vibration of the sample stage can cause disturbance or ripples in the immersion media that can lead to significant distortion in the images. A robust alternative to the use of mechanical scanning stages is a remote focusing system that allows both the objective and the sample to be stationary. One such solution is the employment of a tunable-focus liquid lens in conjunction with a microscope objective to achieve axial scanning through a sample being imaged. Our work demonstrates the implementation of a robust, cost-effective and energy-efficient axial tuning solution for 3D microscopy based on thermo-responsive hydrogel-based tunable liquid lenses.

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM).

PubMed

Wang, Yilin; Kanchanawong, Pakorn

2016-12-01

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and > 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Evaluation of a New Automated Processing System (TACAS™ Pro) for Liquid-Based Procedures.

PubMed

Kuramoto, Hiroyuki; Sugimoto, Naoko; Iwami, Yoshiko; Kato, Chizuyo; Hori, Masuko; Iida, Manichi

2015-01-01

To evaluate a fully automated processing system (TACAS™ Pro) for liquid-based procedures (LBPs). Materials were 3,483 and additionally 502 specimens that were taken at Kanagawa Health Service Association. Specimens obtained with a Cervex-Brush® were first smeared to glass slides using one side of the brush and then processed to TACAS Pro. (1) The microscopy watching time per normal case was 3.65 ± 0.85 min in the conventional procedure, whereas in the LBP it was 1.95 ± 0.60 min, and the latter reduced workload to 53%. (2) The handling time of TACAS Pro per day was 2 h and 25.8 min. The workload at a laboratory offset it and revealed the work saving to be 63.8%. (3) Unsatisfactory rates were 0% in the conventional procedure, whereas in the LBP it was 1.88% at first. The latter rate decreased to 0.5% after system improvement. (4) Specimens which may disturb microscopy analysis were found in 1.06%, including 3 cases of possible carry-over of cells to the following slides. An additional study with the revised system confirmed no carry-over. (5) Incidences of abnormal cytology were consistent between the two methods. The revised automated processing system TACAS Pro is a feasible and useful LBP and reduces the workload of cytology laboratories. © 2015 S. Karger AG, Basel.

Suitability of holographic beam scanning in high resolution applications

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

High-resolution 3D imaging of polymerized photonic crystals by lab-based x-ray nanotomography with 50-nm resolution

NASA Astrophysics Data System (ADS)

Yin, Leilei; Chen, Ying-Chieh; Gelb, Jeff; Stevenson, Darren M.; Braun, Paul A.

2010-09-01

High resolution x-ray computed tomography is a powerful non-destructive 3-D imaging method. It can offer superior resolution on objects that are opaque or low contrast for optical microscopy. Synchrotron based x-ray computed tomography systems have been available for scientific research, but remain difficult to access for broader users. This work introduces a lab-based high-resolution x-ray nanotomography system with 50nm resolution in absorption and Zernike phase contrast modes. Using this system, we have demonstrated high quality 3-D images of polymerized photonic crystals which have been analyzed for band gap structures. The isotropic volumetric data shows excellent consistency with other characterization results.

A high throughput spectral image microscopy system

NASA Astrophysics Data System (ADS)

Gesley, M.; Puri, R.

2018-01-01

A high throughput spectral image microscopy system is configured for rapid detection of rare cells in large populations. To overcome flow cytometry rates and use of fluorophore tags, a system architecture integrates sample mechanical handling, signal processors, and optics in a non-confocal version of light absorption and scattering spectroscopic microscopy. Spectral images with native contrast do not require the use of exogeneous stain to render cells with submicron resolution. Structure may be characterized without restriction to cell clusters of differentiation.

Confocal microscopy as a useful approach to describe gill rakers of Asian species of carp and native filter-feeding fishes of the upper Mississippi River system

USGS Publications Warehouse

Liza R. Walleser,; D.R. Howard,; Sandheinrich, Mark B.; Gaikowski, Mark P.; Amberg, Jon J.

2014-01-01

To better understand potential diet overlap among exotic Asian species of carp and native species of filter-feeding fishes of the upper Mississippi River system, microscopy was used to document morphological differences in the gill rakers. Analysing samples first with light microscopy and subsequently with confocal microscopy, the three-dimensional structure of gill rakers in Hypophthalmichthys molitrix,Hypophthalmichthys nobilis and Dorosoma cepedianum was more thoroughly described and illustrated than previous work with traditional microscopy techniques. The three-dimensional structure of gill rakers in Ictiobus cyprinellus was described and illustrated for the first time.

Accessible Microscopy Workstation for Students and Scientists with Mobility Impairments

ERIC Educational Resources Information Center

Duerstock, Bradley S.

2006-01-01

An integrated accessible microscopy workstation was designed and developed to allow persons with mobility impairments to control all aspects of light microscopy with minimal human assistance. This system, named AccessScope, is capable of performing brightfield and fluorescence microscopy, image analysis, and tissue morphometry requisite for…

A macroscopic non-destructive testing system based on the cantilever-sample contact resonance

NASA Astrophysics Data System (ADS)

Fu, Ji; Lin, Lizhi; Zhou, Xilong; Li, Yingwei; Li, Faxin

2012-12-01

Detecting the inside or buried defects in materials and structures is always a challenge in the field of nondestructive testing (NDT). In this paper, enlightened by the operation principle of the contact resonance force microscopy or atomic force acoustic microscopy (AFAM), we proposed a macroscopic NDT system based on contact resonance of the cantilever-sample surface to detect the local stiffness variations in materials or structures. We fabricated a piezoelectric unimorph with the dimension typically of 150 mm × 8 mm × 2 mm to act as a macroscopic cantilever, whose flexural mode vibration was driven by a wideband power amplifier together with a signal generator. The vibration signal of the macroscopic cantilever is detected by a high sensitive strain gauge bonded on the cantilever surface which is much more stable than the laser diode sensor in AFAM, thus making it very suitable for outdoor operations. Scanning is realized by a three-dimensional motorized stage with the Z axis for pressing force setting. The whole system is controlled by a LabVIEW-based homemade software. Like the AFAM, this NDT system can also work in two modes, i.e., the single-frequency mode and the resonance-tracking mode. In the latter mode, the contact stiffness at each pixel of the sample can be obtained by using the measured contact resonance frequency and a beam dynamics model. Testing results of this NDT system on a grid structure with an opaque panel show that in both modes the prefabricated defect beneath the panel can be detected and the grid structures can be clearly "seen," which indicates the validity of this NDT system. The sensitivity of this NDT system was also examined.

On-Chip Titration of an Anticoagulant Argatroban and Determination of the Clotting Time within Whole Blood or Plasma Using a Plug-Based Microfluidic System

PubMed Central

Song, Helen; Li, Hung-Wing; Munson, Matthew S.; Van Ha, Thuong G.; Ismagilov, Rustem F.

2006-01-01

This paper describes extending plug-based microfluidics to handling complex biological fluids such as blood, solving the problem of injecting additional reagents into plugs, and applying this system to measuring of clotting time in small volumes of whole blood and plasma. Plugs are droplets transported through microchannels by fluorocarbon fluids. A plug-based microfluidic system was developed to titrate an anticoagulant (argatroban) into blood samples and to measure the clotting time using the activated partial thromboplastin time (APTT) test. To carry out these experiments, the following techniques were developed for a plug-based system: (i) using Teflon AF coating on the microchannel wall to enable formation of plugs containing blood and transport of the solid fibrin clots within plugs, (ii) using a hydrophilic glass capillary to enable reliable merging of a reagent from an aqueous stream into plugs, (iii) using bright-field microscopy to detect the formation of a fibrin clot within plugs and using fluorescent microscopy to detect the production of thrombin using a fluorogenic substrate, and (iv) titration of argatroban (0–1.5 μg/mL) into plugs and measurement of the resulting APTTs at room temperature (23 °C) and physiological temperature (37 °C). APTT measurements were conducted with normal pooled plasma (platelet-poor plasma) and with donor’s blood samples (both whole blood and platelet-rich plasma). APTT values and APTT ratios measured by the plug-based microfluidic device were compared to the results from a clinical laboratory at 37 °C. APTT obtained from the on-chip assay were about double those from the clinical laboratory but the APTT ratios from these two methods agreed well with each other. PMID:16841902

Electron Microscopy.

ERIC Educational Resources Information Center

Beer, Michael

1980-01-01

Reviews technical aspects of structure determination in biological electron microscopy (EM). Discusses low dose EM, low temperature microscopy, electron energy loss spectra, determination of mass or molecular weight, and EM of labeled systems. Cites 34 references. (CS)

Image recombination transform algorithm for superresolution structured illumination microscopy

PubMed Central

Zhou, Xing; Lei, Ming; Dan, Dan; Yao, Baoli; Yang, Yanlong; Qian, Jia; Chen, Guangde; Bianco, Piero R.

2016-01-01

Abstract. Structured illumination microscopy (SIM) is an attractive choice for fast superresolution imaging. The generation of structured illumination patterns made by interference of laser beams is broadly employed to obtain high modulation depth of patterns, while the polarizations of the laser beams must be elaborately controlled to guarantee the high contrast of interference intensity, which brings a more complex configuration for the polarization control. The emerging pattern projection strategy is much more compact, but the modulation depth of patterns is deteriorated by the optical transfer function of the optical system, especially in high spatial frequency near the diffraction limit. Therefore, the traditional superresolution reconstruction algorithm for interference-based SIM will suffer from many artifacts in the case of projection-based SIM that possesses a low modulation depth. Here, we propose an alternative reconstruction algorithm based on image recombination transform, which provides an alternative solution to address this problem even in a weak modulation depth. We demonstrated the effectiveness of this algorithm in the multicolor superresolution imaging of bovine pulmonary arterial endothelial cells in our developed projection-based SIM system, which applies a computer controlled digital micromirror device for fast fringe generation and multicolor light-emitting diodes for illumination. The merit of the system incorporated with the proposed algorithm allows for a low excitation intensity fluorescence imaging even less than 1  W/cm2, which is beneficial for the long-term, in vivo superresolved imaging of live cells and tissues. PMID:27653935

Sensitive and selective cataluminescence-based sensor system for acetone and diethyl ether determination.

PubMed

Wang, Qihui; Li, Bo; Wang, Yuhuai; Shou, Zhouxiang; Shi, Guolong

2015-05-01

A three-dimensional hierarchical CdO nanostructure with a novel bio-inspired morphology is reported. The field emission scanning electronic microscopy, transmission electron microscopy and X-ray diffractometer were employed to characterize the as-prepared samples. In gas-sensing measurements, acetone and diethyl ether were employed as target gases to investigate cataluminescence (CTL) sensing properties of the CdO nanostructure. The results show that the as-fabricated CdO nanostructure exhibited outstanding CTL properties such as stable intensity, high signal/noise values, short response and recovery time. The limit of detection of acetone and diethyl ether was ca. 6.5 ppm and 6.7 ppm, respectively, which was below the standard permitted concentrations. Additionally, a principal components analysis method was used to investigate the recognizable ability of the CTL sensor, and it was found that acetone and diethyl ether can be distinguished clearly. The performance of the bio-inspired CdO nanostructure-based sensor system suggested the promising application of the CdO nanostructure as a novel highly efficient CTL sensing material. Copyright © 2014 John Wiley & Sons, Ltd.

Multimodal computational microscopy based on transport of intensity equation

NASA Astrophysics Data System (ADS)

Li, Jiaji; Chen, Qian; Sun, Jiasong; Zhang, Jialin; Zuo, Chao

2016-12-01

Transport of intensity equation (TIE) is a powerful tool for phase retrieval and quantitative phase imaging, which requires intensity measurements only at axially closely spaced planes without a separate reference beam. It does not require coherent illumination and works well on conventional bright-field microscopes. The quantitative phase reconstructed by TIE gives valuable information that has been encoded in the complex wave field by passage through a sample of interest. Such information may provide tremendous flexibility to emulate various microscopy modalities computationally without requiring specialized hardware components. We develop a requisite theory to describe such a hybrid computational multimodal imaging system, which yields quantitative phase, Zernike phase contrast, differential interference contrast, and light field moment imaging, simultaneously. It makes the various observations for biomedical samples easy. Then we give the experimental demonstration of these ideas by time-lapse imaging of live HeLa cell mitosis. Experimental results verify that a tunable lens-based TIE system, combined with the appropriate postprocessing algorithm, can achieve a variety of promising imaging modalities in parallel with the quantitative phase images for the dynamic study of cellular processes.

Stripe artifact elimination based on nonsubsampled contourlet transform for light sheet fluorescence microscopy

NASA Astrophysics Data System (ADS)

Liang, Xiao; Zang, Yali; Dong, Di; Zhang, Liwen; Fang, Mengjie; Yang, Xin; Arranz, Alicia; Ripoll, Jorge; Hui, Hui; Tian, Jie

2016-10-01

Stripe artifacts, caused by high-absorption or high-scattering structures in the illumination light path, are a common drawback in both unidirectional and multidirectional light sheet fluorescence microscopy (LSFM), significantly deteriorating image quality. To circumvent this problem, we present an effective multidirectional stripe remover (MDSR) method based on nonsubsampled contourlet transform (NSCT), which can be used for both unidirectional and multidirectional LSFM. In MDSR, a fast Fourier transform (FFT) filter is designed in the NSCT domain to shrink the stripe components and eliminate the noise. Benefiting from the properties of being multiscale and multidirectional, MDSR succeeds in eliminating stripe artifacts in both unidirectional and multidirectional LSFM. To validate the method, MDSR has been tested on images from a custom-made unidirectional LSFM system and a commercial multidirectional LSFM system, clearly demonstrating that MDSR effectively removes most of the stripe artifacts. Moreover, we performed a comparative experiment with the variational stationary noise remover and the wavelet-FFT methods and quantitatively analyzed the results with a peak signal-to-noise ratio, showing an improved noise removal when using the MDSR method.

Video-rate functional photoacoustic microscopy at depths

NASA Astrophysics Data System (ADS)

Wang, Lidai; Maslov, Konstantin; Xing, Wenxin; Garcia-Uribe, Alejandro; Wang, Lihong V.

2012-10-01

We report the development of functional photoacoustic microscopy capable of video-rate high-resolution in vivo imaging in deep tissue. A lightweight photoacoustic probe is made of a single-element broadband ultrasound transducer, a compact photoacoustic beam combiner, and a bright-field light delivery system. Focused broadband ultrasound detection provides a 44-μm lateral resolution and a 28-μm axial resolution based on the envelope (a 15-μm axial resolution based on the raw RF signal). Due to the efficient bright-field light delivery, the system can image as deep as 4.8 mm in vivo using low excitation pulse energy (28 μJ per pulse, 0.35 mJ/cm2 on the skin surface). The photoacoustic probe is mounted on a fast-scanning voice-coil scanner to acquire 40 two-dimensional (2-D) B-scan images per second over a 9-mm range. High-resolution anatomical imaging is demonstrated in the mouse ear and brain. Via fast dual-wavelength switching, oxygen dynamics of mouse cardio-vasculature is imaged in realtime as well.

Fiber-optic-bundle-based optical coherence tomography.

PubMed

Xie, Tuqiang; Mukai, David; Guo, Shuguang; Brenner, Matthew; Chen, Zhongping

2005-07-15

A fiber-optic-bundle-based optical coherence tomography (OCT) probe method is presented. The experimental results demonstrate this multimode optical fiber-bundle-based OCT system can achieve a lateral resolution of 12 microm and an axial resolution of 10 microm with a superluminescent diode source. This novel OCT imaging approach eliminates any moving parts in the probe and has a primary advantage for use in extremely compact and safe OCT endoscopes for imaging internal organs and great potential to be combined with confocal endoscopic microscopy.

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.

Novel nanocarriers for topical drug delivery: investigating delivery efficiency and distribution in skin using two-photon microscopy

NASA Astrophysics Data System (ADS)

Kirejev, Vladimir; Guldbrand, Stina; Bauer, Brigitte; Smedh, Maria; Ericson, Marica B.

2011-03-01

The complex structure of skin represents an effective barrier against external environmental factors, as for example, different chemical and biochemical compounds, yeast, bacterial and viral infections. However, this impermeability prevents efficient transdermal drug delivery which limits the number of drugs that are able to penetrate the skin efficiently. Current trends in drug application through skin focus on the design and use of nanocarriers for transport of active compounds. The transport systems applied so far have several drawbacks, as they often have low payload, high toxicity, a limited variability of inclusion molecules, or long degradation times. The aim of these current studies is to investigate novel topical drug delivery systems, e.g. nanocarriers based on cyclic oligosaccharides - cyclodextrins (CD) or iron (III)-based metal-organic frameworks (MOF). Earlier studies on cell cultures imply that these drug nanocarriers show promising characteristics compared to other drug delivery systems. In our studies, we use two-photon microscopy to investigate the ability of the nanocarriers to deliver compounds through ex-vivo skin samples. Using near infrared light for excitation in the so called optical window of skin allows deep-tissue visualization of drug distribution and localization. In addition, it is possible to employ two-photon based fluorescence correlation spectroscopy for quantitative analysis of drug distribution and concentrations in different cell layers.

Cellulose Perversions

PubMed Central

Canejo, João P.; Godinho, Maria H.

2013-01-01

Cellulose micro/nano-fibers can be produced by electrospinning from liquid crystalline solutions. Scanning electron microscopy (SEM), as well as atomic force microscopy (AFM) and polarizing optical microscopy (POM) measurements showed that cellulose-based electrospun fibers can curl and twist, due to the presence of an off-core line defect disclination, which was present when the fibers were prepared. This permits the mimicking of the shapes found in many systems in the living world, e.g., the tendrils of climbing plants, three to four orders of magnitude larger. In this work, we address the mechanism that is behind the spirals’ and helices’ appearance by recording the trajectories of the fibers toward diverse electrospinning targets. The intrinsic curvature of the system occurs via asymmetric contraction of an internal disclination line, which generates different shrinkages of the material along the fiber. The completely different instabilities observed for isotropic and anisotropic electrospun solutions at the exit of the needle seem to corroborate the hypothesis that the intrinsic curvature of the material is acquired during liquid crystalline sample processing inside the needle. The existence of perversions, which joins left and right helices, is also investigated by using suspended, as well as flat, targets. Possible routes of application inspired from the living world are addressed. PMID:28809215

High speed multiphoton imaging

NASA Astrophysics Data System (ADS)

Li, Yongxiao; Brustle, Anne; Gautam, Vini; Cockburn, Ian; Gillespie, Cathy; Gaus, Katharina; Lee, Woei Ming

2016-12-01

Intravital multiphoton microscopy has emerged as a powerful technique to visualize cellular processes in-vivo. Real time processes revealed through live imaging provided many opportunities to capture cellular activities in living animals. The typical parameters that determine the performance of multiphoton microscopy are speed, field of view, 3D imaging and imaging depth; many of these are important to achieving data from in-vivo. Here, we provide a full exposition of the flexible polygon mirror based high speed laser scanning multiphoton imaging system, PCI-6110 card (National Instruments) and high speed analog frame grabber card (Matrox Solios eA/XA), which allows for rapid adjustments between frame rates i.e. 5 Hz to 50 Hz with 512 × 512 pixels. Furthermore, a motion correction algorithm is also used to mitigate motion artifacts. A customized control software called Pscan 1.0 is developed for the system. This is then followed by calibration of the imaging performance of the system and a series of quantitative in-vitro and in-vivo imaging in neuronal tissues and mice.

Active Metal Brazing and Adhesive Bonding of Titanium to C/C Composites for Heat Rejection System

NASA Technical Reports Server (NTRS)

Singh, M.; Shpargel, Tarah; Cerny, Jennifer

2006-01-01

Robust assembly and integration technologies are critically needed for the manufacturing of heat rejection system (HRS) components for current and future space exploration missions. Active metal brazing and adhesive bonding technologies are being assessed for the bonding of titanium to high conductivity Carbon-Carbon composite sub components in various shapes and sizes. Currently a number of different silver and copper based active metal brazes and adhesive compositions are being evaluated. The joint microstructures were examined using optical microscopy, and scanning electron microscopy (SEM) coupled with energy dispersive spectrometry (EDS). Several mechanical tests have been employed to ascertain the effectiveness of different brazing and adhesive approaches in tension and in shear that are both simple and representative of the actual system and relatively straightforward in analysis. The results of these mechanical tests along with the fractographic analysis will be discussed. In addition, advantages, technical issues and concerns in using different bonding approaches will also be presented.

Application of a Loop-Type Laboratory Biofilm Reactor to the Evaluation of Biofilm for Some Metallic Materials and Polymers such as Urinary Stents and Catheters.

PubMed

Kanematsu, Hideyuki; Kudara, Hikonaru; Kanesaki, Shun; Kogo, Takeshi; Ikegai, Hajime; Ogawa, Akiko; Hirai, Nobumitsu

2016-10-11

A laboratory biofilm reactor (LBR) was modified to a new loop-type closed system in order to evaluate novel stents and catheter materials using 3D optical microscopy and Raman spectroscopy. Two metallic specimens, pure nickel and cupronickel (80% Cu-20% Ni), along with two polymers, silicone and polyurethane, were chosen as examples to ratify the system. Each set of specimens was assigned to the LBR using either tap water or an NB (Nutrient broth based on peptone from animal foods and beef extract mainly)-cultured solution with E-coli formed over 48-72 h. The specimens were then analyzed using Raman Spectroscopy. 3D optical microscopy was employed to corroborate the Raman Spectroscopy results for only the metallic specimens since the inherent roughness of the polymer specimens made such measurements difficult. The findings suggest that the closed loop-type LBR together with Raman spectroscopy analysis is a useful method for evaluating biomaterials as a potential urinary system.

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping.

PubMed

Roth, Gary A; Sosa Peña, Maria del Pilar; Neu-Baker, Nicole M; Tahiliani, Sahil; Brenner, Sara A

2015-12-08

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Impact of solvents and supercritical CO2 drying on the morphology and structure of polymer-based biofilms

NASA Astrophysics Data System (ADS)

Causa, Andrea; Salerno, Aurelio; Domingo, Concepción; Acierno, Domenico; Filippone, Giovanni

2014-05-01

In the present work, two-dimensional systems based on biodegradable polymers such as poly(ɛ-caprolactone) (PCL), poly(ethylene oxide) (PEO) and polylactic acid (PLA) are fabricated by means of a sustainable approach which consists in inducing phase separation in solutions of such polymers and "green" solvents, namely ethyl lactate (EL) and ethyl acetate (EA). The extraction of the solvent is promoted by a controlled drying process, which is performed in either air or supercritical CO2. The latter can indeed act as both an antisolvent, which favors the deposition of the polymer by forming a mixture with EL and EA, and a plasticizing agent, whose solvation and transport properties may considerably affect the microstructure and crystallinity of the polymer films. The morphological, topographical and crystalline properties of the films are tailored through a judicial selection of the materials and the processing conditions and assessed by means of thermal analyses, polarized optical microscopy, scanning electron microscopy and confocal interferometric microscopy. The results show that the morphological and crystalline properties of the films are strongly dependent on the choice of both the polymer/solvent system and the operating conditions during the drying step. In particular, the morphological, topographical and thermal properties of films prepared starting from highly crystalline polymers, namely PCL and PEO, are greatly affected by the crystallization of the material. Conversely, the less crystalline PLA forms almost completely amorphous films.

Interstitial-phase precipitation in iron-base alloys: a comparative study

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

Pelton, A.R.

1982-06-01

Recent developments have elucidated the atomistic mechanisms of precipitation of interstitial elements in simple alloy systems. However, in the more technologically important iron base alloys, interstitial phase precipitation is generally not well understood. The present experimental study was therefore designed to test the applicability of these concepts to more complex ferrous alloys. Hence, a comparative study was made of interstitial phase precipitation in ferritic Fe-Si-C and in austenitic phosphorus-containing Fe-Cr-Ni steels. These systems were subjected to a variety of quench-age thermal treatments, and the microstructural development was subsequently characterized by transmission electron microscopy.

Microencapsulation of Polyfunctional Amines for Self-Healing of Epoxy-Based Composites

DTIC Science & Technology

2008-01-01

MICROENCAPSULATION OF POLYFUNCTIONAL AMINES FOR SELF-HEALING OF EPOXY-BASED COMPOSITES David A. McIlroy*§, Ben J. Blaiszik,¥ Paul V. Braun... microcapsules containing an amine hardener (DEH-52, Dow Chemical) for use as the hardener in a 2-part epoxy healing system consisting of epoxy...microscope. Scanning electron microscopy was performed on a Philips XL30 ESEM-FEG instrument. Microencapsulation Procedure. 10 g of a 2:1 v/v

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

PubMed

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

2016-05-16

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

Rapid on-site detection of airborne asbestos fibers and potentially hazardous nanomaterials using fluorescence microscopy-based biosensing.

PubMed

Kuroda, Akio; Alexandrov, Maxym; Nishimura, Tomoki; Ishida, Takenori

2016-06-01

A large number of peptides with binding affinity to various inorganic materials have been identified and used as linkers, catalysts, and building blocks in nanotechnology and nanobiotechnology. However, there have been few applications of material-binding peptides in the fluorescence microscopy-based biosensing (FM method) of environmental pollutants. A notable exception is the application of the FM method for the detection of asbestos, a dangerous industrial toxin that is still widely used in many developing countries. This review details the selection and isolation of asbestos-binding proteins and peptides with sufficient specificity to distinguish asbestos from a large variety of safer fibrous materials used as asbestos substitutes. High sensitivity to nanoscale asbestos fibers (30-35 nm in diameter) invisible under conventional phase contrast microscopy can be achieved. The FM method is the basis for developing an automated system for asbestos biosensing that can be used for on-site testing with a portable fluorescence microscope. In the future, the FM method could also become a useful tool for detecting other potentially hazardous nanomaterials in the environment. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Brain tumor classification using AFM in combination with data mining techniques.

PubMed

Huml, Marlene; Silye, René; Zauner, Gerald; Hutterer, Stephan; Schilcher, Kurt

2013-01-01

Although classification of astrocytic tumors is standardized by the WHO grading system, which is mainly based on microscopy-derived, histomorphological features, there is great interobserver variability. The main causes are thought to be the complexity of morphological details varying from tumor to tumor and from patient to patient, variations in the technical histopathological procedures like staining protocols, and finally the individual experience of the diagnosing pathologist. Thus, to raise astrocytoma grading to a more objective standard, this paper proposes a methodology based on atomic force microscopy (AFM) derived images made from histopathological samples in combination with data mining techniques. By comparing AFM images with corresponding light microscopy images of the same area, the progressive formation of cavities due to cell necrosis was identified as a typical morphological marker for a computer-assisted analysis. Using genetic programming as a tool for feature analysis, a best model was created that achieved 94.74% classification accuracy in distinguishing grade II tumors from grade IV ones. While utilizing modern image analysis techniques, AFM may become an important tool in astrocytic tumor diagnosis. By this way patients suffering from grade II tumors are identified unambiguously, having a less risk for malignant transformation. They would benefit from early adjuvant therapies.

Solving the inverse scattering problem in reflection-mode dynamic speckle-field phase microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zhou, Renjie; So, Peter T. C.; Yaqoob, Zahid; Jin, Di; Hosseini, Poorya; Kuang, Cuifang; Singh, Vijay Raj; Kim, Yang-Hyo; Dasari, Ramachandra R.

2017-02-01

Most of the quantitative phase microscopy systems are unable to provide depth-resolved information for measuring complex biological structures. Optical diffraction tomography provides a non-trivial solution to it by 3D reconstructing the object with multiple measurements through different ways of realization. Previously, our lab developed a reflection-mode dynamic speckle-field phase microscopy (DSPM) technique, which can be used to perform depth resolved measurements in a single shot. Thus, this system is suitable for measuring dynamics in a layer of interest in the sample. DSPM can be also used for tomographic imaging, which promises to solve the long-existing "missing cone" problem in 3D imaging. However, the 3D imaging theory for this type of system has not been developed in the literature. Recently, we have developed an inverse scattering model to rigorously describe the imaging physics in DSPM. Our model is based on the diffraction tomography theory and the speckle statistics. Using our model, we first precisely calculated the defocus response and the depth resolution in our system. Then, we further calculated the 3D coherence transfer function to link the 3D object structural information with the axially scanned imaging data. From this transfer function, we found that in the reflection mode excellent sectioning effect exists in the low lateral spatial frequency region, thus allowing us to solve the "missing cone" problem. Currently, we are working on using this coherence transfer function to reconstruct layered structures and complex cells.

Lab-On-Chip Clinorotation System for Live-Cell Microscopy Under Simulated Microgravity

NASA Technical Reports Server (NTRS)

Yew, Alvin G.; Atencia, Javier; Chinn, Ben; Hsieh, Adam H.

2013-01-01

Cells in microgravity are subject to mechanical unloading and changes to the surrounding chemical environment. How these factors jointly influence cellular function is not well understood. We can investigate their role using ground-based analogues to spaceflight, where mechanical unloading is simulated through the time-averaged nullification of gravity. The prevailing method for cellular microgravity simulation is to use fluid-filled containers called clinostats. However, conventional clinostats are not designed for temporally tracking cell response, nor are they able to establish dynamic fluid environments. To address these needs, we developed a Clinorotation Time-lapse Microscopy (CTM) system that accommodates lab-on- chip cell culture devices for visualizing time-dependent alterations to cellular behavior. For the purpose of demonstrating CTM, we present preliminary results showing time-dependent differences in cell area between human mesenchymal stem cells (hMSCs) under modeled microgravity and normal gravity.

Lab-On-Chip Clinorotation System for Live-Cell Microscopy Under Simulated Microgravity

NASA Technical Reports Server (NTRS)

Yew, Alvin G.; Atencia, Javier; Chinn, Ben; Hsieh, Adam H.

1980-01-01

Cells in microgravity are subject to mechanical unloading and changes to the surrounding chemical environment. How these factors jointly influence cellular function is not well understood. We can investigate their role using ground-based analogues to spaceflight, where mechanical unloading is simulated through the time-averaged nullification of gravity. The prevailing method for cellular microgravity simulation is to use fluid-filled containers called clinostats. However, conventional clinostats are not designed for temporally tracking cell response, nor are they able to establish dynamic fluid environments. To address these needs, we developed a Clinorotation Time-lapse Microscopy (CTM) system that accommodates lab-on- chip cell culture devices for visualizing time-dependent alterations to cellular behavior. For the purpose of demonstrating CTM, we present preliminary results showing time-dependent differences in cell area between human mesenchymal stem cells (hMSCs) under modeled microgravity and normal gravity.

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera

NASA Astrophysics Data System (ADS)

Cruz Perez, Carlos; Lauri, Antonella; Symvoulidis, Panagiotis; Cappetta, Michele; Erdmann, Arne; Westmeyer, Gil Gregor

2015-09-01

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera.

PubMed

Perez, Carlos Cruz; Lauri, Antonella; Symvoulidis, Panagiotis; Cappetta, Michele; Erdmann, Arne; Westmeyer, Gil Gregor

2015-09-01

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.

Light sensitive polymer obtained by dispersion of azo-functionalized POSS nanoparticles

NASA Astrophysics Data System (ADS)

Miniewicz, A.; Tomkowicz, M.; Karpinski, P.; Sznitko, L.; Mossety-Leszczak, B.; Dutkiewicz, M.

2015-07-01

Hybrid inorganic-organic nanoparticles based on cubic siloxane cage (RSiO3/2)8, known as polyhedral oligosilsesquioxane (POSS), have been functionalized by eight groups of azo-benzene mesogens and dispersed in poly(methyl methacrylate) PMMA matrix. Presence of azo-benzene units adds an important light-driven functionality to the system due to their photoisomerization resulting in refractive index and/or absorption changes of the whole system. The polymer films containing various concentrations of azo-POSS nanoparticles show remarkable changes of surface morphology being either transparent (at low POSS concentration) or highly scattering (at high POSS concentration) for visible light. Surface structures were examined by optical microscopy as well as by atomic force microscopy (AFM). Results of photoinduced alignment are discussed in the framework of light-induced modification of the aliphatic chains containing azo-benzene photoisomerizing moieties and self-organization process.

Multi-Algorithm Particle Simulations with Spatiocyte.

PubMed

Arjunan, Satya N V; Takahashi, Koichi

2017-01-01

As quantitative biologists get more measurements of spatially regulated systems such as cell division and polarization, simulation of reaction and diffusion of proteins using the data is becoming increasingly relevant to uncover the mechanisms underlying the systems. Spatiocyte is a lattice-based stochastic particle simulator for biochemical reaction and diffusion processes. Simulations can be performed at single molecule and compartment spatial scales simultaneously. Molecules can diffuse and react in 1D (filament), 2D (membrane), and 3D (cytosol) compartments. The implications of crowded regions in the cell can be investigated because each diffusing molecule has spatial dimensions. Spatiocyte adopts multi-algorithm and multi-timescale frameworks to simulate models that simultaneously employ deterministic, stochastic, and particle reaction-diffusion algorithms. Comparison of light microscopy images to simulation snapshots is supported by Spatiocyte microscopy visualization and molecule tagging features. Spatiocyte is open-source software and is freely available at http://spatiocyte.org .

Enabling Interactive Measurements from Large Coverage Microscopy

PubMed Central

Bajcsy, Peter; Vandecreme, Antoine; Amelot, Julien; Chalfoun, Joe; Majurski, Michael; Brady, Mary

2017-01-01

Microscopy could be an important tool for characterizing stem cell products if quantitative measurements could be collected over multiple spatial and temporal scales. With the cells changing states over time and being several orders of magnitude smaller than cell products, modern microscopes are already capable of imaging large spatial areas, repeat imaging over time, and acquiring images over several spectra. However, characterizing stem cell products from such large image collections is challenging because of data size, required computations, and lack of interactive quantitative measurements needed to determine release criteria. We present a measurement web system consisting of available algorithms, extensions to a client-server framework using Deep Zoom, and the configuration know-how to provide the information needed for inspecting the quality of a cell product. The cell and other data sets are accessible via the prototype web-based system at http://isg.nist.gov/deepzoomweb. PMID:28663600

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles.

PubMed

Seynhaeve, Ann L B; Ten Hagen, Timo L M

2017-11-01

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Fe-Based Amorphous Coatings on AISI 4130 Structural Steel for Corrosion Resistance

NASA Astrophysics Data System (ADS)

Katakam, Shravana; Santhanakrishnan, S.; Dahotre, Narendra B.

2012-06-01

The current study focuses on synthesizing a novel functional coating for corrosion resistance applications, via laser surface alloying. The iron-based (Fe48Cr15Mo14Y2C15B) amorphous precursor powder is used for laser surface alloying on AISI 4130 steel substrate, with a continuous wave ytterbium Nd-YAG fiber laser. The corrosion resistance of the coatings is evaluated for different processing conditions. The microstructural evolution and the response of the microstructure to the corrosive environment is studied using x-ray diffraction, scanning electron microscopy, and transmission electron microscopy. Microstructural studies indicate the presence of face-centered cubic Fe-based dendrites intermixed within an amorphous matrix along with fine crystalline precipitates. The corrosion resistance of the coatings decrease with an increase in laser energy density, which is attributed to the precipitation and growth of chromium carbide. The enhanced corrosion resistance of the coatings processed with low energy density is attributed to the self-healing mechanism of this amorphous system.

Nanopositioning for polarimetric characterization.

PubMed

Qureshi, Naser; Kolokoltsev, Oleg V; Ortega-Martínez, Roberto; Ordoñez-Romero, C L

2008-12-01

A positioning system with approximately nanometer resolution has been developed based on a new implementation of a motor-driven screw scheme. In contrast to conventional positioning systems based on piezoelectric elements, this system shows remarkably low levels of drift and vibration, and eliminates the need for position feedback during typical data acquisition processes. During positioning or scanning processes, non-repeatability and hysteresis problems inherent in mechanical positioning systems are greatly reduced using a software feedback scheme. As a result, we are able to demonstrate an average mechanical resolution of 1.45 nm and near diffraction-limited imaging using scanning optical microscopy. We propose this approach to nanopositioning as a readily accessible alternative enabling high spatial resolution scanning probe characterization (e.g., polarimetry) and provide practical details for its implementation.

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).

Towards fully automated Identification of Vesicle-Membrane Fusion Events in TIRF Microscopy

NASA Astrophysics Data System (ADS)

Vallotton, Pascal; James, David E.; Hughes, William E.

2007-11-01

Total Internal Reflection Fluorescence Microscopy (TIRFM) is imposing itself as the tool of choice for studying biological activity in close proximity to the plasma membrane. For example, the exquisite selectivity of TIRFM allows monitoring the diffusion of GFP-phogrin vesicles and their recruitment to the plasma membrane in pancreatic β-cells. We present a novel computer vision system for automatically identifying the elusive fusion events of GFP-phogrin vesicles with the plasma membrane. Our method is based on robust object tracking and matched filtering. It should accelerate the quantification of TIRFM data and allow the extraction of more biological information from image data to support research in diabetes and obesity.

Scanning gate microscopy of quantum rings: effects of an external magnetic field and of charged defects.

PubMed

Pala, M G; Baltazar, S; Martins, F; Hackens, B; Sellier, H; Ouisse, T; Bayot, V; Huant, S

2009-07-01

We study scanning gate microscopy (SGM) in open quantum rings obtained from buried semiconductor InGaAs/InAlAs heterostructures. By performing a theoretical analysis based on the Keldysh-Green function approach we interpret the radial fringes observed in experiments as the effect of randomly distributed charged defects. We associate SGM conductance images with the local density of states (LDOS) of the system. We show that such an association cannot be made with the current density distribution. By varying an external magnetic field we are able to reproduce recursive quasi-classical orbits in LDOS and conductance images, which bear the same periodicity as the Aharonov-Bohm effect.

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

NASA Astrophysics Data System (ADS)

Muckenhirn, Sylvain

2016-03-01

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

A simple and low-cost structured illumination microscopy using a pico-projector

NASA Astrophysics Data System (ADS)

Özgürün, Baturay

2018-02-01

Here, development of a low-cost structured illumination microscopy (SIM) based on a pico-projector is presented. The pico-projector consists of independent red, green and blue LEDs that remove need for an external illumination source. Moreover, display element of the pico-projector serves as a pattern generating spatial light modulator. A simple lens group is employed to couple light from the projector to an epi-illumination port of a commercial microscope system. 2D sub SIM images are acquired and synthesized to surpass the diffraction limit using 40x (0.75 NA) objective. Resolution of the reconstructed SIM images is verified with a dye-and-object object and a fixed cell sample.

Quantitative optical coherence microscopy for the in situ investigation of the biofilm

NASA Astrophysics Data System (ADS)

Meleppat, Ratheesh Kumar; Shearwood, Christopher; Keey, Seah Leong; Matham, Murukeshan Vadakke

2016-12-01

This paper explores the potential of optical coherence microscopy (OCM) for the in situ monitoring of biofilm growth. The quantitative imaging of the early developmental biology of a representative biofilm, Klebsiella pneumonia (KP-1), was performed using a swept source-based Fourier domain OCM system. The growth dynamics of the KP-1 biofilms and their transient response under perturbation was investigated using the enface visualization of microcolonies and their spatial localization. Furthermore, the optical density (OD) and planar density of the biofilms are calculated using an OCM technique and compared with OD and colony forming units measured using standard procedures via the sampling of the flow-cell effluent.

Simulating contrast inversion in atomic force microscopy imaging with real-space pseudopotentials

NASA Astrophysics Data System (ADS)

Lee, Alex; Sakai, Yuki; Chelikowsky, James

Atomic force microscopy measurements have reported contrast inversions for systems such as Cu2N and graphene that can hamper image interpretation and characterization. Here, we apply a simulation method based on ab initio real-space pseudopotentials to gain an understanding of the tip-sample interactions that influence the inversion. We find that chemically reactive tips induce an attractive binding force that results in the contrast inversion. The inversion is tip height dependent and not observed when using less reactive CO-functionalized tips. Work is supported by the DOE under DOE/DE-FG02-06ER46286 and by the Welch Foundation under Grant F-1837. Computational resources were provided by NERSC and XSEDE.

Memory-effect based deconvolution microscopy for super-resolution imaging through scattering media

NASA Astrophysics Data System (ADS)

Edrei, Eitan; Scarcelli, Giuliano

2016-09-01

High-resolution imaging through turbid media is a fundamental challenge of optical sciences that has attracted a lot of attention in recent years for its wide range of potential applications. Here, we demonstrate that the resolution of imaging systems looking behind a highly scattering medium can be improved below the diffraction-limit. To achieve this, we demonstrate a novel microscopy technique enabled by the optical memory effect that uses a deconvolution image processing and thus it does not require iterative focusing, scanning or phase retrieval procedures. We show that this newly established ability of direct imaging through turbid media provides fundamental and practical advantages such as three-dimensional refocusing and unambiguous object reconstruction.

Memory-effect based deconvolution microscopy for super-resolution imaging through scattering media.

PubMed

Edrei, Eitan; Scarcelli, Giuliano

2016-09-16

High-resolution imaging through turbid media is a fundamental challenge of optical sciences that has attracted a lot of attention in recent years for its wide range of potential applications. Here, we demonstrate that the resolution of imaging systems looking behind a highly scattering medium can be improved below the diffraction-limit. To achieve this, we demonstrate a novel microscopy technique enabled by the optical memory effect that uses a deconvolution image processing and thus it does not require iterative focusing, scanning or phase retrieval procedures. We show that this newly established ability of direct imaging through turbid media provides fundamental and practical advantages such as three-dimensional refocusing and unambiguous object reconstruction.

Joint Research on Scatterometry and AFM Wafer Metrology

NASA Astrophysics Data System (ADS)

Bodermann, Bernd; Buhr, Egbert; Danzebrink, Hans-Ulrich; Bär, Markus; Scholze, Frank; Krumrey, Michael; Wurm, Matthias; Klapetek, Petr; Hansen, Poul-Erik; Korpelainen, Virpi; van Veghel, Marijn; Yacoot, Andrew; Siitonen, Samuli; El Gawhary, Omar; Burger, Sven; Saastamoinen, Toni

2011-11-01

Supported by the European Commission and EURAMET, a consortium of 10 participants from national metrology institutes, universities and companies has started a joint research project with the aim of overcoming current challenges in optical scatterometry for traceable linewidth metrology. Both experimental and modelling methods will be enhanced and different methods will be compared with each other and with specially adapted atomic force microscopy (AFM) and scanning electron microscopy (SEM) measurement systems in measurement comparisons. Additionally novel methods for sophisticated data analysis will be developed and investigated to reach significant reductions of the measurement uncertainties in critical dimension (CD) metrology. One final goal will be the realisation of a wafer based reference standard material for calibration of scatterometers.

Holographic microscopy studies of emulsions

NASA Technical Reports Server (NTRS)

Witherow, W. K.

1981-01-01

A holographic microscopy system that records and observes the dynamic properties of separation of dispersed immiscible fluids is described. The holographic construction system and reconstruction system that were used to obtain particle size and distribution information from the holograms are discussed. The holographic microscopy system is used to observed the phase separating processes in immiscible fluids that were isothermally cooled into the two phase region. Nucleation, growth rates, coalescence, and particle motion are successfully demonstrated with this system. Thus a holographic particle sizing system with a resolution of 2 micrometers and a field of view of 100 cu cm was developed that provides the capability of testing the theories of separating immiscible fluids for particle number densities in the range of 10 to 10 to the 7th power particles.

CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: AXIAL RESOLUTION

EPA Science Inventory

Abstract

Confocal Microscopy System Performance: Axial resolution.
Robert M. Zucker, PhD

Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Re...

Dynamics of intracellular processes in live-cell systems unveiled by fluorescence correlation microscopy.

PubMed

González Bardeci, Nicolás; Angiolini, Juan Francisco; De Rossi, María Cecilia; Bruno, Luciana; Levi, Valeria

2017-01-01

Fluorescence fluctuation-based methods are non-invasive microscopy tools especially suited for the study of dynamical aspects of biological processes. These methods examine spontaneous intensity fluctuations produced by fluorescent molecules moving through the small, femtoliter-sized observation volume defined in confocal and multiphoton microscopes. The quantitative analysis of the intensity trace provides information on the processes producing the fluctuations that include diffusion, binding interactions, chemical reactions and photophysical phenomena. In this review, we present the basic principles of the most widespread fluctuation-based methods, discuss their implementation in standard confocal microscopes and briefly revise some examples of their applications to address relevant questions in living cells. The ultimate goal of these methods in the Cell Biology field is to observe biomolecules as they move, interact with targets and perform their biological action in the natural context. © 2016 IUBMB Life, 69(1):8-15, 2017. © 2016 International Union of Biochemistry and Molecular Biology.

Wavefront sensorless adaptive optics temporal focusing-based multiphoton microscopy

PubMed Central

Chang, Chia-Yuan; Cheng, Li-Chung; Su, Hung-Wei; Hu, Yvonne Yuling; Cho, Keng-Chi; Yen, Wei-Chung; Xu, Chris; Dong, Chen Yuan; Chen, Shean-Jen

2014-01-01

Temporal profile distortions reduce excitation efficiency and image quality in temporal focusing-based multiphoton microscopy. In order to compensate the distortions, a wavefront sensorless adaptive optics system (AOS) was integrated into the microscope. The feedback control signal of the AOS was acquired from local image intensity maximization via a hill-climbing algorithm. The control signal was then utilized to drive a deformable mirror in such a way as to eliminate the distortions. With the AOS correction, not only is the axial excitation symmetrically refocused, but the axial resolution with full two-photon excited fluorescence (TPEF) intensity is also maintained. Hence, the contrast of the TPEF image of a R6G-doped PMMA thin film is enhanced along with a 3.7-fold increase in intensity. Furthermore, the TPEF image quality of 1μm fluorescent beads sealed in agarose gel at different depths is improved. PMID:24940539

A general system for automatic biomedical image segmentation using intensity neighborhoods.

PubMed

Chen, Cheng; Ozolek, John A; Wang, Wei; Rohde, Gustavo K

2011-01-01

Image segmentation is important with applications to several problems in biology and medicine. While extensively researched, generally, current segmentation methods perform adequately in the applications for which they were designed, but often require extensive modifications or calibrations before being used in a different application. We describe an approach that, with few modifications, can be used in a variety of image segmentation problems. The approach is based on a supervised learning strategy that utilizes intensity neighborhoods to assign each pixel in a test image its correct class based on training data. We describe methods for modeling rotations and variations in scales as well as a subset selection for training the classifiers. We show that the performance of our approach in tissue segmentation tasks in magnetic resonance and histopathology microscopy images, as well as nuclei segmentation from fluorescence microscopy images, is similar to or better than several algorithms specifically designed for each of these applications.

Comparison of mechanical characteristics of focused ion beam fabricated silicon nanowires

NASA Astrophysics Data System (ADS)

Ina, Ginnosuke; Fujii, Tatsuya; Kozeki, Takahiro; Miura, Eri; Inoue, Shozo; Namazu, Takahiro

2017-06-01

In this study, we investigate the effects of focused ion beam (FIB)-induced damage and specimen size on the mechanical properties of Si nanowires (NWs) by a microelectromechanical system (MEMS)-based tensile testing technique. By an FIB fabrication technique, three types of Si NWs, which are as-FIB-fabricated, annealed, and FIB-implanted NWs, are prepared. A sacrificial-oxidized NW is also prepared to compare the mechanical properties of these FIB-based NWs. The quasi-static uniaxial tensile tests of all the NWs are conducted by scanning electron microscopy (SEM). The fabrication process and specimen size dependences on Young’s modulus and fracture strength are observed. Annealing is effective for improving the Young’s modulus of the FIB-damaged Si. Transmission electron microscopy (TEM) suggests that the mechanism behind the process dependence on the mechanical characteristics is related to the crystallinity of the FIB-damaged portion.

Repeatability and reproducibility of intracellular molar concentration assessed by synchrotron-based x-ray fluorescence microscopy

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

Merolle, L., E-mail: lucia.merolle@elettra.eu; Gianoncelli, A.; Malucelli, E., E-mail: emil.malucelli@unibo.it

2016-01-28

Elemental analysis of biological sample can give information about content and distribution of elements essential for human life or trace elements whose absence is the cause of abnormal biological function or development. However, biological systems contain an ensemble of cells with heterogeneous chemistry and elemental content; therefore, accurate characterization of samples with high cellular heterogeneity may only be achieved by analyzing single cells. Powerful methods in molecular biology are abundant, among them X-Ray microscopy based on synchrotron light source has gaining increasing attention thanks to its extremely sensitivity. However, reproducibility and repeatability of these measurements is one of the majormore » obstacles in achieving a statistical significance in single cells population analysis. In this study, we compared the elemental content of human colon adenocarcinoma cells obtained by three distinct accesses to synchrotron radiation light.« less

A low-noise measurement system for scanning thermal microscopy resistive nanoprobes based on a transformer ratio-arm bridge

NASA Astrophysics Data System (ADS)

Świątkowski, Michał; Wojtuś, Arkadiusz; Wielgoszewski, Grzegorz; Rudek, Maciej; Piasecki, Tomasz; Jóźwiak, Grzegorz; Gotszalk, Teodor

2018-04-01

Atomic force microscopy (AFM) is a widely used technology for the investigation and characterization of nanomaterials. Its functionality can be easily expanded by applying dedicated extension modules, which can measure the electrical conductivity or temperature of a sample. In this paper, we introduce a transformer ratio-arm bridge setup dedicated to AFM-based thermal imaging. One of the key features of the thermal module is the use of a low-power driving signal that prevents undesirable tip heating during resistance measurement, while the other is the sensor location in a ratio-arm transformer bridge working in the audio frequency range and ensuring galvanic isolation of the tip, enabling contact-mode scanning of electronic circuits. The proposed expansion module is compact and it can be integrated onto the AFM head close to the cantilever. The calibration process and the resolution of 11 mK of the proposed setup are shown.

Varifocal MOEMS fiber scanner for confocal endomicroscopy.

PubMed

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

2014-12-15

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

The method of segmentation of leukocytes in information-measuring systems on the basis of light microscopy

NASA Astrophysics Data System (ADS)

Nikitaev, V. G.; Pronichev, A. N.; Polyakov, E. V.; Zaharenko, Yu V.

2018-01-01

The paper considers the problem of leukocytes segmentation in microscopic images of bone marrow smears for automated diagnosis of the blood system diseases. The method was proposed to solve the problem of segmentation of contacting leukocytes in images of bone marrow smears. The method is based on the analysis of structure of objects of a separation and distances filter in combination with the watershed method and distance transformation method.

1998 Technology Showcase. JOAP International Condition Monitoring Conference.

DTIC Science & Technology

1998-04-01

Systems using Automated SEM/ EDX and New Diagnostic Routines 276 N. W Farrant & T. Luckhurst ADVANCED DIAGNOSTIC SYSTEMS Model-Based Diagnostics of Gas...Microscopy with Energy Dispersive X-Ray (SEM/ EDX ) micro analysis packages and Energy Dispersive X-Ray Fluorescence (EDXRF) analytical equipment. Therqfore...wear particles separated by ferrogram method. a- I WEAR PARTICLE A SLAS 97 (HOME PAGE) Fig I Home Page NONFE;RROUS MATERIAL A wW~ a48 -1, rV fr , ý b

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

NASA Astrophysics Data System (ADS)

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

2005-03-01

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

Liquid crystal alignment in electro-responsive nanostructured thermosetting materials based on block copolymer dispersed liquid crystal.

PubMed

Tercjak, A; Garcia, I; Mondragon, I

2008-07-09

Novel well-defined nanostructured thermosetting systems were prepared by modification of a diglicydylether of bisphenol-A epoxy resin (DGEBA) with 10 or 15 wt% amphiphilic poly(styrene-b-ethylene oxide) block copolymer (PSEO) and 30 or 40 wt% low molecular weight liquid crystal 4'-(hexyl)-4-biphenyl-carbonitrile (HBC) using m-xylylenediamine (MXDA) as a curing agent. The competition between well-defined nanostructured materials and the ability for alignment of the liquid crystal phase in the materials obtained has been studied by atomic and electrostatic force microscopy, AFM and EFM, respectively. Based on our knowledge, this is the first time that addition of an adequate amount (10 wt%) of a block copolymer to 40 wt% HBC-(DGEBA/MXDA) leads to a well-organized nanostructured thermosetting system (between a hexagonal and worm-like ordered structure), which is also electro-responsive with high rate contrast. This behavior was confirmed using electrostatic force microscopy (EFM), by means of the response of the HBC liquid crystal phase to the voltage applied to the EFM tip. In contrast, though materials containing 15 wt% PSEO and 30 wt% HBC also form a well-defined nanostructured thermosetting system, they do not show such a high contrast between the uncharged and charged surface.

Intravital Microscopy Imaging Approaches for Image-Guided Drug Delivery Systems

PubMed Central

Kirui, Dickson K.; Ferrari, Mauro

2016-01-01

Rapid technical advances in the field of non-linear microscopy have made intravital microscopy a vital pre-clinical tool for research and development of imaging-guided drug delivery systems. The ability to dynamically monitor the fate of macromolecules in live animals provides invaluable information regarding properties of drug carriers (size, charge, and surface coating), physiological, and pathological processes that exist between point-of-injection and the projected of site of delivery, all of which influence delivery and effectiveness of drug delivery systems. In this Review, we highlight how integrating intravital microscopy imaging with experimental designs (in vitro analyses and mathematical modeling) can provide unique information critical in the design of novel disease-relevant drug delivery platforms with improved diagnostic and therapeutic indexes. The Review will provide the reader an overview of the various applications for which intravital microscopy has been used to monitor the delivery of diagnostic and therapeutic agents and discuss some of their potential clinical applications. PMID:25901526

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

Nucleoside-Lipid-Based Nanocarriers for Sorafenib Delivery

NASA Astrophysics Data System (ADS)

Benizri, Sebastien; Ferey, Ludivine; Alies, Bruno; Mebarek, Naila; Vacher, Gaelle; Appavoo, Ananda; Staedel, Cathy; Gaudin, Karen; Barthélémy, Philippe

2018-01-01

Although the application of sorafenib, a small inhibitor of tyrosine protein kinases, to cancer treatments remains a worldwide option in chemotherapy, novel strategies are needed to address the low water solubility (< 5 μM), toxicity, and side effects issues of this drug. In this context, the use of nanocarriers is currently investigated in order to overcome these drawbacks. In this contribution, we report a new type of sorafenib-based nanoparticles stabilized by hybrid nucleoside-lipids. The solid lipid nanoparticles (SLNs) showed negative or positive zeta potential values depending on the nucleoside-lipid charge. Transmission electron microscopy of sorafenib-loaded SLNs revealed parallelepiped nanoparticles of about 200 nm. Biological studies achieved on four different cell lines, including liver and breast cancers, revealed enhanced anticancer activities of Sorafenib-based SLNs compared to the free drug. Importantly, contrast phase microscopy images recorded after incubation of cancer cells in the presence of SLNs at high concentration in sorafenib (> 80 μM) revealed a total cancer cell death in all cases. These results highlight the potential of nucleoside-lipid-based SLNs as drug delivery systems.

Applications and challenges of digital pathology and whole slide imaging.

PubMed

Higgins, C

2015-07-01

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

Investigating the performance of reconstruction methods used in structured illumination microscopy as a function of the illumination pattern's modulation frequency

NASA Astrophysics Data System (ADS)

Shabani, H.; Sánchez-Ortiga, E.; Preza, C.

2016-03-01

Surpassing the resolution of optical microscopy defined by the Abbe diffraction limit, while simultaneously achieving optical sectioning, is a challenging problem particularly for live cell imaging of thick samples. Among a few developing techniques, structured illumination microscopy (SIM) addresses this challenge by imposing higher frequency information into the observable frequency band confined by the optical transfer function (OTF) of a conventional microscope either doubling the spatial resolution or filling the missing cone based on the spatial frequency of the pattern when the patterned illumination is two-dimensional. Standard reconstruction methods for SIM decompose the low and high frequency components from the recorded low-resolution images and then combine them to reach a high-resolution image. In contrast, model-based approaches rely on iterative optimization approaches to minimize the error between estimated and forward images. In this paper, we study the performance of both groups of methods by simulating fluorescence microscopy images from different type of objects (ranging from simulated two-point sources to extended objects). These simulations are used to investigate the methods' effectiveness on restoring objects with various types of power spectrum when modulation frequency of the patterned illumination is changing from zero to the incoherent cut-off frequency of the imaging system. Our results show that increasing the amount of imposed information by using a higher modulation frequency of the illumination pattern does not always yield a better restoration performance, which was found to be depended on the underlying object. Results from model-based restoration show performance improvement, quantified by an up to 62% drop in the mean square error compared to standard reconstruction, with increasing modulation frequency. However, we found cases for which results obtained with standard reconstruction methods do not always follow the same trend.

Efficient protein immobilization on polyethersolfone electrospun nanofibrous membrane via covalent binding for biosensing applications.

PubMed

Mahmoudifard, Matin; Soudi, Sara; Soleimani, Masoud; Hosseinzadeh, Simzar; Esmaeili, Elaheh; Vossoughi, Manouchehr

2016-01-01

In this paper we introduce novel strategy for antibody immobilization using high surface area electrospun nanofibrous membrane based on ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling chemistry. To present the high performance of proposed biosensors, anti-staphylococcus enterotoxin B (anti-SEB) was used as a model to demonstrate the utility of our proposed system. Polymer solution of polyethersolfone was used to fabricate fine nanofibrous membrane. Moreover, industrial polyvinylidene fluoride membrane and conventional microtiter plate were also used to compare the efficiency of antibody immobilization. Scanning electron microscopy images were taken to study the morphology of the membranes. The surface activation of nanofibrous membrane was done with the help of O2 plasma. PES nanofibrous membrane with carboxyl functional groups for covalent attachment of antibodies were treated by EDC/NHS coupling agent. The quantity of antibody immobilization was measured by enzyme-linked immuno sorbent assay (ELISA) method. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy was performed to confirm the covalent immobilization of antibody on membrane. Atomic force microscopy, scanning electron microscopy and invert fluorescence microscopy were used to analyze the antibody distribution pattern on solid surfaces. Results show that oxygen plasma treatment effectively increased the amount of antibody immobilization through EDC/NHS coupling chemistry. It was found that the use of nanofibrous membrane causes the improved detection signal of ELISA based biosensors in comparison to the standard assay carried out in the 96-well microtiter plate. This method has the potential to improve the ELISA-based biosensor and we believe that this technique can be used in various biosensing methods. Copyright © 2015. Published by Elsevier B.V.

Controlled poorly soluble drug release from solid self-microemulsifying formulations with high viscosity hydroxypropylmethylcellulose.

PubMed

Yi, Tao; Wan, Jiangling; Xu, Huibi; Yang, Xiangliang

2008-08-07

The objective of this work was the development of a controlled release system based on self-microemulsifying mixture aimed for oral delivery of poorly water-soluble drugs. HPMC-based particle formulations were prepared by spray drying containing a model drug (nimodipine) of low water solubility and hydroxypropylmethylcellulose (HPMC) of high viscosity. One type of formulations contained nimodipine mixed with HPMC and the other type of formulations contained HPMC and nimodipine dissolved in a self-microemulsifying system (SMES) consisting of ethyl oleate, Cremophor RH 40 and Labrasol. Based on investigation by transmission electron microscopy (TEM), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and X-ray powder diffraction, differences were found in the particle structure between both types of formulations. In vitro release was performed and characterized by the power law. Nimodipine release from both types of formulations showed a controlled release profile and the two power law parameters, n and K, correlated to the viscosity of HPMC. The parameters were also influenced by the presence of SMES. For the controlled release solid SMES, oil droplets containing dissolved nimodipine diffused out of HPMC matrices following exposure to aqueous media. Thus, it is possible to control the in vitro release of poorly soluble drugs from solid oral dosage forms containing SMES.

Phases and interfaces from real space atomically resolved data: Physics-based deep data image analysis

DOE PAGES

Vasudevan, Rama K.; Ziatdinov, Maxim; Jesse, Stephen; ...

2016-08-12

Advances in electron and scanning probe microscopies have led to a wealth of atomically resolved structural and electronic data, often with ~1–10 pm precision. However, knowledge generation from such data requires the development of a physics-based robust framework to link the observed structures to macroscopic chemical and physical descriptors, including single phase regions, order parameter fields, interfaces, and structural and topological defects. Here, we develop an approach based on a synergy of sliding window Fourier transform to capture the local analog of traditional structure factors combined with blind linear unmixing of the resultant 4D data set. This deep data analysismore » is ideally matched to the underlying physics of the problem and allows reconstruction of the a priori unknown structure factors of individual components and their spatial localization. We demonstrate the principles of this approach using a synthetic data set and further apply it for extracting chemical and physically relevant information from electron and scanning tunneling microscopy data. Furthermore, this method promises to dramatically speed up crystallographic analysis in atomically resolved data, paving the road toward automatic local structure–property determinations in crystalline and quasi-ordered systems, as well as systems with competing structural and electronic order parameters.« less

NiAl Oxidation Reaction Processes Studied In Situ Using MEMS-Based Closed-Cell Gas Reaction Transmission Electron Microscopy

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

Unocic, Kinga A.; Shin, Dongwon; Unocic, Raymond R.

The nanoscale oxidation mechanisms and kinetics of a model β-NiAl system were investigated using in situ closed-cell gas reaction scanning transmission electron microscopy (STEM). Here, we directly visualize the dynamic structural and chemical changes that occur during high-temperature oxidation at a high spatial resolution of 50.3Ni–49.7Al (at.%) nanoparticles under static air conditions at 730 Torr with heating up to 750 °C at 5 °C/s. A MEMS-based gas cell system, with microfabricated heater devices and a gas delivery system, was used to reveal site-specific oxidation initiation sites. Through time-resolved annular dark-field STEM imaging, we tracked the nanoscale oxidation kinetics of Almore » 2O 3. After oxidation at 750 °C, nucleation of voids at the Ni/Al 2O 3 interface was observed along a NiAl grain boundary, followed by the formation of faceted NiO crystals. Small faceted cubic crystals of NiO were formed at the initial stage of oxidation at high PO 2 due to the outward self-diffusion of Ni 2+ ions, followed by the formation of a mixture of metastable and stable α-Al 2O 3 at the oxide/metal interface that is attributed to a PO 2 decrease with oxidation time, which agreed with thermodynamic modeling calculations. Furthermore, the results from these in situ oxidation experiments in the β-NiAl system are in agreement with the established oxidation mechanisms; however, with in situ closed-cell gas microscopy it is now feasible to investigate nanoscale oxidation mechanisms and kinetics in real time and at high spatial resolution and can be broadly applied to understand the basic high-temperature oxidation mechanisms for a wide range of alloy compositions.« less

NiAl Oxidation Reaction Processes Studied In Situ Using MEMS-Based Closed-Cell Gas Reaction Transmission Electron Microscopy

DOE PAGES

Unocic, Kinga A.; Shin, Dongwon; Unocic, Raymond R.; ...

2017-02-07

The nanoscale oxidation mechanisms and kinetics of a model β-NiAl system were investigated using in situ closed-cell gas reaction scanning transmission electron microscopy (STEM). Here, we directly visualize the dynamic structural and chemical changes that occur during high-temperature oxidation at a high spatial resolution of 50.3Ni–49.7Al (at.%) nanoparticles under static air conditions at 730 Torr with heating up to 750 °C at 5 °C/s. A MEMS-based gas cell system, with microfabricated heater devices and a gas delivery system, was used to reveal site-specific oxidation initiation sites. Through time-resolved annular dark-field STEM imaging, we tracked the nanoscale oxidation kinetics of Almore » 2O 3. After oxidation at 750 °C, nucleation of voids at the Ni/Al 2O 3 interface was observed along a NiAl grain boundary, followed by the formation of faceted NiO crystals. Small faceted cubic crystals of NiO were formed at the initial stage of oxidation at high PO 2 due to the outward self-diffusion of Ni 2+ ions, followed by the formation of a mixture of metastable and stable α-Al 2O 3 at the oxide/metal interface that is attributed to a PO 2 decrease with oxidation time, which agreed with thermodynamic modeling calculations. Furthermore, the results from these in situ oxidation experiments in the β-NiAl system are in agreement with the established oxidation mechanisms; however, with in situ closed-cell gas microscopy it is now feasible to investigate nanoscale oxidation mechanisms and kinetics in real time and at high spatial resolution and can be broadly applied to understand the basic high-temperature oxidation mechanisms for a wide range of alloy compositions.« less

Mayo Clinic/Renal Pathology Society Consensus Report on Pathologic Classification, Diagnosis, and Reporting of GN.

PubMed

Sethi, Sanjeev; Haas, Mark; Markowitz, Glen S; D'Agati, Vivette D; Rennke, Helmut G; Jennette, J Charles; Bajema, Ingeborg M; Alpers, Charles E; Chang, Anthony; Cornell, Lynn D; Cosio, Fernando G; Fogo, Agnes B; Glassock, Richard J; Hariharan, Sundaram; Kambham, Neeraja; Lager, Donna J; Leung, Nelson; Mengel, Michael; Nath, Karl A; Roberts, Ian S; Rovin, Brad H; Seshan, Surya V; Smith, Richard J H; Walker, Patrick D; Winearls, Christopher G; Appel, Gerald B; Alexander, Mariam P; Cattran, Daniel C; Casado, Carmen Avila; Cook, H Terence; De Vriese, An S; Radhakrishnan, Jai; Racusen, Lorraine C; Ronco, Pierre; Fervenza, Fernando C

2016-05-01

Renal pathologists and nephrologists met on February 20, 2015 to establish an etiology/pathogenesis-based system for classification and diagnosis of GN, with a major aim of standardizing the kidney biopsy report of GN. On the basis of etiology/pathogenesis, GN is classified into the following five pathogenic types, each with specific disease entities: immune-complex GN, pauci-immune GN, antiglomerular basement membrane GN, monoclonal Ig GN, and C3 glomerulopathy. The pathogenesis-based classification forms the basis of the kidney biopsy report. To standardize the report, the diagnosis consists of a primary diagnosis and a secondary diagnosis. The primary diagnosis should include the disease entity/pathogenic type (if disease entity is not known) followed in order by pattern of injury (mixed patterns may be present); score/grade/class for disease entities, such as IgA nephropathy, lupus nephritis, and ANCA GN; and additional features as detailed herein. A pattern diagnosis as the sole primary diagnosis is not recommended. Secondary diagnoses should be reported separately and include coexisting lesions that do not form the primary diagnosis. Guidelines for the report format, light microscopy, immunofluorescence microscopy, electron microscopy, and ancillary studies are also provided. In summary, this consensus report emphasizes a pathogenesis-based classification of GN and provides guidelines for the standardized reporting of GN. Copyright © 2016 by the American Society of Nephrology.

Environmental scanning electron microscopy gold immunolabeling in cell biology.

PubMed

Rosso, Francesco; Papale, Ferdinando; Barbarisi, Alfonso

2013-01-01

Immunogold labeling (IGL) technique has been utilized by many authors in combination with scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to obtain the identification/localization of receptors and antigens, both in cells and tissues. Environmental scanning electron microscopy (ESEM) represents an important tool in biomedical research, since it does not require any severe processing of the sample, lowering the risk of generating artifacts and interfere with the IGL procedure. The absence of metal coating could yield further advantages for our purpose as the labeling detection is based on the atomic number difference between nanogold spheres and the biological material. Using the gaseous secondary electron detector, compositional contrast is easily revealed by the backscattered electron component of the signal. In spite of this fact, only few published papers present a combination of ESEM and IGL. Hereby we present our method, optimized to improve the intensity and the specificity of the labeling signal, in order to obtain a semiquantitative evaluation of the labeling signal.In particular, we used a combination of IGL and ESEM to detect the presence of a protein on the cell surface. To achieve this purpose, we chose as an experimental system 3T3 Swiss albino mouse fibroblasts and galectin-3.

Simple approach to three-color two-photon microscopy by a fiber-optic wavelength convertor.

PubMed

Li, Kuen-Che; Huang, Lynn L H; Liang, Jhih-Hao; Chan, Ming-Che

2016-11-01

A simple approach to multi-color two-photon microscopy of the red, green, and blue fluorescent indicators was reported based on an ultra-compact 1.03-μm femtosecond laser and a nonlinear fiber. Inside the nonlinear fiber, the 1.03-μm laser pulses were simultaneously blue-shifted to 0.6~0.8 μm and red-shifted to 1.2~1.4 μm region by the Cherenkov radiation and fiber Raman gain effects. The wavelength-shifted 0.6~0.8 μm and 1.2~1.4 μm radiations were co-propagated with the residual non-converted 1.03-μm pulses inside the same nonlinear fiber to form a fiber-output three-color femtosecond source. The application of the multi-wavelength sources on multi-color two-photon fluorescence microscopy were also demonstrated. Overall, due to simple system configuration, convenient wavelength conversion, easy wavelength tunability within the entire 0.7~1.35 μm bio-penetration window and less requirement for high power and bulky light sources, the simple approach to multi-color two-photon microscopy could be widely applicable as an easily implemented and excellent research tool for future biomedical and possibly even clinical applications.

High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

NASA Astrophysics Data System (ADS)

Regmi, Raju; Mohan, Kavya; Mondal, Partha Pratim

2014-09-01

Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro- to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine.

Frequency division multiplexed multi-color fluorescence microscope system

NASA Astrophysics Data System (ADS)

Le, Vu Nam; Yang, Huai Dong; Zhang, Si Chun; Zhang, Xin Rong; Jin, Guo Fan

2017-10-01

Grayscale camera can only obtain gray scale image of object, while the multicolor imaging technology can obtain the color information to distinguish the sample structures which have the same shapes but in different colors. In fluorescence microscopy, the current method of multicolor imaging are flawed. Problem of these method is affecting the efficiency of fluorescence imaging, reducing the sampling rate of CCD etc. In this paper, we propose a novel multiple color fluorescence microscopy imaging method which based on the Frequency division multiplexing (FDM) technology, by modulating the excitation lights and demodulating the fluorescence signal in frequency domain. This method uses periodic functions with different frequency to modulate amplitude of each excitation lights, and then combine these beams for illumination in a fluorescence microscopy imaging system. The imaging system will detect a multicolor fluorescence image by a grayscale camera. During the data processing, the signal obtained by each pixel of the camera will be processed with discrete Fourier transform, decomposed by color in the frequency domain and then used inverse discrete Fourier transform. After using this process for signals from all of the pixels, monochrome images of each color on the image plane can be obtained and multicolor image is also acquired. Based on this method, this paper has constructed and set up a two-color fluorescence microscope system with two excitation wavelengths of 488 nm and 639 nm. By using this system to observe the linearly movement of two kinds of fluorescent microspheres, after the data processing, we obtain a two-color fluorescence dynamic video which is consistent with the original image. This experiment shows that the dynamic phenomenon of multicolor fluorescent biological samples can be generally observed by this method. Compared with the current methods, this method can obtain the image signals of each color at the same time, and the color video's frame rate is consistent with the frame rate of the camera. The optical system is simpler and does not need extra color separation element. In addition, this method has a good filtering effect on the ambient light or other light signals which are not affected by the modulation process.

Improving your four-dimensional image: traveling through a decade of light-sheet-based fluorescence microscopy research.

PubMed

Strobl, Frederic; Schmitz, Alexander; Stelzer, Ernst H K

2017-06-01

Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.

SISGR: Room Temperature Single-Molecule Detection and Imaging by Stimulated Emission Microscopy

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

Xie, Xiaoliang Sunney

Single-molecule spectroscopy has made considerable impact on many disciplines including chemistry, physics, and biology. To date, most single-molecule spectroscopy work is accomplished by detecting fluorescence. On the other hand, many naturally occurring chromophores, such as retinal, hemoglobin and cytochromes, do not have detectable fluorescence. There is an emerging need for single-molecule spectroscopy techniques that do not require fluorescence. In the last proposal period, we have successfully demonstrated stimulated emission microscopy, single molecule absorption, and stimulated Raman microscopy based on a high-frequency modulation transfer technique. These first-of-a- kind new spectroscopy/microscopy methods tremendously improved our ability to observe molecules that fluorescence weakly,more » even to the limit of single molecule detection for absorption measurement. All of these methods employ two laser beams: one (pump beam) excites a single molecule to a real or virtual excited state, and the other (probe beam) monitors the absorption/emission property of the single. We extract the intensity change of the probe beam with high sensitivity by implementing a high-frequency phase-sensitive detection scheme, which offers orders of magnitude improvement in detection sensitivity over direct absorption/emission measurement. However, single molecule detection based on fluorescence or absorption is fundamentally limited due to their broad spectral response. It is important to explore other avenues in single molecule detection and imaging which provides higher molecular specificity for studying a wide variety of heterogeneous chemical and biological systems. This proposal aimed to achieve single-molecule detection sensitivity with near resonance stimulated Raman scattering (SRS) microscopy. SRS microscopy was developed in our lab as a powerful technique for imaging heterogeneous samples based on their intrinsic vibrational contrasts, which provides much higher molecular specificity than absorption and fluorescence. Current sensitivity limit of SRS microscopy has not yet reached single molecule detection. We proposed to capitalize on our state-of-the-art SRS microscopy and develop near-resonance enhanced SRS for single molecule detection of carotenoids and heme proteins. The specific aims we pursued are: (1) building the next SRS generation microscope that utilizes near resonance enhancement to allow detection and imaging of single molecules with undetectable fluorescence, such as -carotene. (2) using near-resonance SRS as a contrast mechanism to study dye-sensitize semiconductor interface, elucidating the heterogeneous electron ejection kinetics with high spatial and temporal resolution. (3) studying the binding and unbinding of oxygen in single hemoglobin molecules in order to gain molecular level understanding of the long-standing issue of cooperativity. The new methods developed in the fund period of this grant have advanced the detection sensitivity in many aspects. Near-resonance SRS improved the signal by using shorter wavelengths for SRS microscopy. Frequency modulation and multi-color SRS target the reduction of background to improve the chemical specificity of SRS while maintaining the high imaging speed. Time-domain coherent Raman scattering microscopy targets to reduce the noise floor of coherent Raman microscopy. These methods have already demonstrated first-of-a-kind new applications in biology and medical research. However, we are still one order of magnitude away from single molecule limit. It is important to continue to improve the laser specification and develop new imaging methods to finally achieve label-free single molecule microscopy.« less

CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: QA TESTS, QUANTITATION AND SPECTROSCOPY

EPA Science Inventory

Confocal Microscopy System Performance: QA tests, Quantitation and Spectroscopy.

Robert M. Zucker 1 and Jeremy M. Lerner 2,
1Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research Development, U.S. Environmen...

EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: PRETTY PICTURES OR CONFOCAL QA

EPA Science Inventory

Evaluation of confocal microscopy system performance: Pretty pictures or confocal QA?

Robert M. Zucker

Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, N...

A portable microscopy system for fluorescence, polarized, and brightfield imaging

NASA Astrophysics Data System (ADS)

Gordon, Paul; Wattinger, Rolla; Lewis, Cody; Venancio, Vinicius Paula; Mertens-Talcott, Susanne U.; Coté, Gerard

2018-02-01

The use of mobile phones to conduct diagnostic microscopy at the point-of-care presents intriguing possibilities for the advancement of high-quality medical care in remote settings. However, it is challenging to create a single device that can adapt to the ever-varying camera technologies in phones or that can image with the customization that multiple modalities require for applications such as malaria diagnosis. A portable multi-modal microscope system is presented that utilizes a Raspberry Pi to collect and transmit data wirelessly to a myriad of electronic devices for image analysis. The microscopy system is capable of providing to the user correlated brightfield, polarized, and fluorescent images of samples fixed on traditional microscopy slides. The multimodal diagnostic capabilities of the microscope were assessed by measuring parasitemia of Plasmodium falciparum-infected thin blood smears. The device is capable of detecting fluorescently-labeled DNA using FITC excitation (490 nm) and emission (525 nm), the birefringent P. falciparum byproduct hemozoin, and detecting brightfield absorption with a resolution of 0.78 micrometers (element 9-3 of a 1951 Air Force Target). This microscopy system is a novel portable imaging tool that may be a viable candidate for field implementation if challenges of system durability, cost considerations, and full automation can be overcome.

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.

Neural plasticity explored by correlative two-photon and electron/SPIM microscopy

NASA Astrophysics Data System (ADS)

Allegra Mascaro, A. L.; Silvestri, L.; Costantini, I.; Sacconi, L.; Maco, B.; Knott, G. W.; Pavone, F. S.

2013-06-01

Plasticity of the central nervous system is a complex process which involves the remodeling of neuronal processes and synaptic contacts. However, a single imaging technique can reveal only a small part of this complex machinery. To obtain a more complete view, complementary approaches should be combined. Two-photon fluorescence microscopy, combined with multi-photon laser nanosurgery, allow following the real-time dynamics of single neuronal processes in the cerebral cortex of living mice. The structural rearrangement elicited by this highly confined paradigm of injury can be imaged in vivo first, and then the same neuron could be retrieved ex-vivo and characterized in terms of ultrastructural features of the damaged neuronal branch by means of electron microscopy. Afterwards, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, based on the use of major blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron imaged in living mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting from its apical portion, the whole pyramidal neuron can then be segmented and located in the correct cortical layer. With the correlative approach presented here, researchers will be able to place in a three-dimensional anatomic context the neurons whose dynamics have been observed with high detail in vivo.

Digital microscopy. Bringing new technology into focus.

PubMed

2010-06-01

Digital microscopy enables the scanning of microscope slides so that they can be viewed, analyzed, and archived on a computer. While the technology is not yet widely accepted by pathologists, a switch to digital microscopy systems seems to be inevitable in the near future.

Growth and microstructural investigation of multiwall carbon nanotubes fabricated using electrodeposited nickel nanodeposits and chemical vapor deposition method

NASA Astrophysics Data System (ADS)

Zanganeh, Navid; Rajabi, Armin; Torabi, Morteza; Allahkarami, Masoud; Moghaddas, Arshak; Sadrnezhaad, S. K.

2014-09-01

This study proposes a common approach for growing multiwall carbon nanotubes (MWCNTs) on nickel nanodeposits. MWCNT growth was performed in two separate stages. In the first stage, nickel nanodeposits were electrodeposited on n-Si(1 1 1):H substrate in the presence of sulfuric acid. Based on atomic force microscopy (AFM) observations, the nickel deposits had a fairly polygonal morphology and were distributed on the prepared n-Si(1 1 1):H substrate. In the second stage, acetylene gas was decomposed on the surfaces of the nickel nanodeposits using chemical vapor deposition method at 700 °C. When carbon is saturated in a catalyst, it acts as a primary nucleating element for MWCNT growth. The structure of the MWCNTs was also investigated using scanning electron microscopy, high-resolution transmission electron microscopy, X-ray diffraction, and Raman spectroscopy. Results showed that the synthesized MWCNTs had a small wall thickness and were formed under the experimental conditions applied to the system.

MULTISCALE TENSOR ANISOTROPIC FILTERING OF FLUORESCENCE MICROSCOPY FOR DENOISING MICROVASCULATURE.

PubMed

Prasath, V B S; Pelapur, R; Glinskii, O V; Glinsky, V V; Huxley, V H; Palaniappan, K

2015-04-01

Fluorescence microscopy images are contaminated by noise and improving image quality without blurring vascular structures by filtering is an important step in automatic image analysis. The application of interest here is to automatically extract the structural components of the microvascular system with accuracy from images acquired by fluorescence microscopy. A robust denoising process is necessary in order to extract accurate vascular morphology information. For this purpose, we propose a multiscale tensor with anisotropic diffusion model which progressively and adaptively updates the amount of smoothing while preserving vessel boundaries accurately. Based on a coherency enhancing flow with planar confidence measure and fused 3D structure information, our method integrates multiple scales for microvasculature preservation and noise removal membrane structures. Experimental results on simulated synthetic images and epifluorescence images show the advantage of our improvement over other related diffusion filters. We further show that the proposed multiscale integration approach improves denoising accuracy of different tensor diffusion methods to obtain better microvasculature segmentation.

Far-field nanoscopy on a semiconductor quantum dot via a rapid-adiabatic-passage-based switch

NASA Astrophysics Data System (ADS)

Kaldewey, Timo; Kuhlmann, Andreas V.; Valentin, Sascha R.; Ludwig, Arne; Wieck, Andreas D.; Warburton, Richard J.

2018-02-01

The diffraction limit prevents a conventional optical microscope from imaging at the nanoscale. However, nanoscale imaging of molecules is possible by exploiting an intensity-dependent molecular switch1-3. This switch is translated into a microscopy scheme, stimulated emission depletion microscopy4-7. Variants on this scheme exist3,8-13, yet all exploit an incoherent response to the lasers. We present a scheme that relies on a coherent response to a laser. Quantum control of a two-level system proceeds via rapid adiabatic passage, an ideal molecular switch. We implement this scheme on an ensemble of quantum dots. Each quantum dot results in a bright spot in the image with extent down to 30 nm (λ/31). There is no significant loss of intensity with respect to confocal microscopy, resulting in a factor of 10 improvement in emitter position determination. The experiments establish rapid adiabatic passage as a versatile tool in the super-resolution toolbox.

Note: Tormenta: An open source Python-powered control software for camera based optical microscopy.

PubMed

Barabas, Federico M; Masullo, Luciano A; Stefani, Fernando D

2016-12-01

Until recently, PC control and synchronization of scientific instruments was only possible through closed-source expensive frameworks like National Instruments' LabVIEW. Nowadays, efficient cost-free alternatives are available in the context of a continuously growing community of open-source software developers. Here, we report on Tormenta, a modular open-source software for the control of camera-based optical microscopes. Tormenta is built on Python, works on multiple operating systems, and includes some key features for fluorescence nanoscopy based on single molecule localization.

Note: Tormenta: An open source Python-powered control software for camera based optical microscopy

NASA Astrophysics Data System (ADS)

Barabas, Federico M.; Masullo, Luciano A.; Stefani, Fernando D.

2016-12-01

Until recently, PC control and synchronization of scientific instruments was only possible through closed-source expensive frameworks like National Instruments' LabVIEW. Nowadays, efficient cost-free alternatives are available in the context of a continuously growing community of open-source software developers. Here, we report on Tormenta, a modular open-source software for the control of camera-based optical microscopes. Tormenta is built on Python, works on multiple operating systems, and includes some key features for fluorescence nanoscopy based on single molecule localization.

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

Insights into the prominent effect of mahanimbine on Acanthamoeba castellanii: Cell profiling analysis based on microscopy techniques

NASA Astrophysics Data System (ADS)

Hashim, Fatimah; Amin, Nakisah Mat

2017-02-01

Mahanimbine (MH), has been shown to have antiamoeba properties. Therefore, the aim of this study was to assess the growth inhibitory mechanisms of MH on Acanthamoeba castellanii, a causative agents for Acanthamoeba keratitis. The IC50 value obtained for MH against A. castellanii was 1.18 µg/ml. Light and scanning electron microscopy observation showed that most cells were in cystic appearance. While transmission electron microscopy observation revealed changes at the ultrastructural level and fluorescence microscopy observation indicated the induction of apoptosis and autophagic activity in the amoeba cytoplasms. In conclusion, MH has very potent anti-amoebic properties on A. castellanii as is shown by cytotoxicity analyses based on microscopy techniques.

Corrosion of Nickel-Based Alloys in Ultra-High Temperature Heat Transfer Fluid

NASA Astrophysics Data System (ADS)

Wang, Tao; Reddy, Ramana G.

2017-03-01

MgCl2-KCl binary system has been proposed to be used as high temperature reactor coolant. Due to its relatively low melting point, good heat capacity and excellent thermal stability, this system can also be used in high operation temperature concentrating solar power generation system as heat transfer fluid (HTF). The corrosion behaviors of nickel based alloys in MgCl2-KCl molten salt system at 1,000 °C were determined based on long-term isothermal dipping test. After 500 h exposure tests under strictly maintained high purity argon gas atmosphere, the weight loss and corrosion rate analysis were conducted. Among all the tested samples, Ni-201 demonstrated the lowest corrosion rate due to the excellent resistance of Ni to high temperature element dissolution. Detailed surface topography and corrosion mechanisms were also determined by using scanning electron microscopy (SEM) equipped with energy dispersive spectrometer (EDS).

Lensless microscopy technique for static and dynamic colloidal systems.

PubMed

Alvarez-Palacio, D C; Garcia-Sucerquia, J

2010-09-15

We present the application of a lensless microscopy technique known as digital in-line holographic microscopy (DIHM) to image dynamic and static colloidal systems of microspheres. DIHM has been perfected up to the point that submicrometer lateral resolution with several hundreds of micrometers depth of field is achieved with visible light; it is shown that the lateral resolution of DIHM is enough to resolve self-assembled colloidal monolayers built up from polystyrene spheres with submicrometer diameters. The time resolution of DIHM is of the order of 4 frames/s at 2048 x 2048 pixels, which represents an overall improvement of 16 times the time resolution of confocal scanning microscopy. This feature is applied to the visualization of the migration of dewetting fronts in dynamic colloidal systems and the formation of front-like arrangements of particles. Copyright 2010 Elsevier Inc. All rights reserved.

A semi-automated, field-portable microscopy platform for clinical diagnostic applications

NASA Astrophysics Data System (ADS)

Jagannadh, Veerendra Kalyan; Srinivasan, Rajesh; Gorthi, Sai Siva

2015-08-01

Clinical microscopy is a versatile diagnostic platform used for diagnosis of a multitude of diseases. In the recent past, many microfluidics based point-of-care diagnostic devices have been developed, which serve as alternatives to microscopy. However, these point-of-care devices are not as multi-functional and versatile as clinical microscopy. With the use of custom designed optics and microfluidics, we have developed a versatile microscopy-based cellular diagnostic platform, which can be used at the point of care. The microscopy platform presented here is capable of detecting infections of very low parasitemia level (in a very small quantity of sample), without the use of any additional computational hardware. Such a cost-effective and portable diagnostic device, would greatly impact the quality of health care available to people living in rural locations of the world. Apart from clinical diagnostics, it's applicability to field research in environmental microbiology has also been outlined.

Convective-diffusion-based fluorescence correlation spectroscopy for detection of a trace amount of E. coli in water.

PubMed

Qing, De-Kui; Mengüç, M Pinar; Payne, Fred A; Danao, Mary-Grace C

2003-06-01

Fluorescence correlation spectroscopy (FCS) is adapted for a new procedure to detect trace amounts of Escherichia coli in water. The present concept is based on convective diffusion rather than Brownian diffusion and employs confocal microscopy as in traditional FCS. With this system it is possible to detect concentrations as small as 1.5 x 10(5) E. coli per milliliter (2.5 x 10(-16) M). This concentration corresponds to an approximately 1.0-nM level of Rhodamine 6G dyes. A detailed analysis of the optical system is presented, and further improvements for the procedure are discussed.

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.

Turning Microscopy in the Medical Curriculum Digital: Experiences from The Faculty of Health and Medical Sciences at University of Copenhagen

PubMed Central

Vainer, Ben; Mortensen, Niels Werner; Poulsen, Steen Seier; Sørensen, Allan Have; Olsen, Jørgen; Saxild, Hans Henrik; Johansen, Flemming Fryd

2017-01-01

Familiarity with the structure and composition of normal tissue and an understanding of the changes that occur during disease is pivotal to the study of the human body. For decades, microscope slides have been central to teaching pathology in medical courses and related subjects at the University of Copenhagen. Students had to learn how to use a microscope and envisage three-dimensional processes that occur in the body from two-dimensional glass slides. Here, we describe how a PathXL virtual microscopy system for teaching pathology and histology at the Faculty has recently been implemented, from an administrative, an economic, and a teaching perspective. This fully automatic digital microscopy system has been received positively by both teachers and students, and a decision was made to convert all courses involving microscopy to the virtual microscopy format. As a result, conventional analog microscopy will be phased out from the fall of 2016. PMID:28382225

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

PubMed

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

2014-01-01

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

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.

Reagent-free and portable detection of Bacillus anthracis spores using a microfluidic incubator and smartphone microscope

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

Hutchison, Janine R.; Erikson, Rebecca L.; Sheen, Allison M.

Rapid, cost-effective bacterial detection systems are needed to respond to potential biothreat events. Here we report the use of smartphone-based microscopy in combination with a simple microfluidic incubation device to detect 5000 Bacillus anthracis spores in 3 hours. This field-deployable approach is compatible with real-time PCR for secondary confirmation.

A Phase Separation Route to Synthesize α-Fe2O3 Porous Nanofibers via Electrospinning for Ultrafast Ethanol Sensing

NASA Astrophysics Data System (ADS)

Dong, Shuwen; Yan, Shuang; Gao, Wenyuan; Liu, Guishan; Hao, Hongshun

2018-07-01

A facile and economic procedure was provided to synthesize α-Fe2O3 nanofibers. In this procedure, porous α-Fe2O3 nanofibers were obtained by a single-polymer/binary-solvent system, while solid α-Fe2O3 nanofibers were prepared by a single-polymer/single-solvent system. The crystal structure and morphology of both samples were characterized by x-ray diffraction, scanning electron microscopy, transmission electron microscopy and nitrogen adsorption/desorption isotherms. The formation mechanism of porous structure was based on solvent evaporation-induced phase separation by the use of mixed solvents with different volatility. Furthermore, ethanol-sensing performance of the porous α-Fe2O3 nanofibers was evaluated and compared with solid α-Fe2O3 nanofibers. Results from gas-sensing measurements reveal that porous α-Fe2O3 nanofibers exhibit higher sensitivity and slightly longer recovery time than solid α-Fe2O3 nanofibers. Over all, the gas sensor based on porous α-Fe2O3 nanofibers shows excellent ethanol-sensing capability with high sensitivity and ultrafast response/recovery behaviors, indicating its potential application as a real-time monitoring gas sensor.

Digitally controlled analog proportional-integral-derivative (PID) controller for high-speed scanning probe microscopy

NASA Astrophysics Data System (ADS)

Dukic, Maja; Todorov, Vencislav; Andany, Santiago; Nievergelt, Adrian P.; Yang, Chen; Hosseini, Nahid; Fantner, Georg E.

2017-12-01

Nearly all scanning probe microscopes (SPMs) contain a feedback controller, which is used to move the scanner in the direction of the z-axis in order to maintain a constant setpoint based on the tip-sample interaction. The most frequently used feedback controller in SPMs is the proportional-integral (PI) controller. The bandwidth of the PI controller presents one of the speed limiting factors in high-speed SPMs, where higher bandwidths enable faster scanning speeds and higher imaging resolution. Most SPM systems use digital signal processor-based PI feedback controllers, which require analog-to-digital and digital-to-analog converters. These converters introduce additional feedback delays which limit the achievable imaging speed and resolution. In this paper, we present a digitally controlled analog proportional-integral-derivative (PID) controller. The controller implementation allows tunability of the PID gains over a large amplification and frequency range, while also providing precise control of the system and reproducibility of the gain parameters. By using the analog PID controller, we were able to perform successful atomic force microscopy imaging of a standard silicon calibration grating at line rates up to several kHz.

Digitally controlled analog proportional-integral-derivative (PID) controller for high-speed scanning probe microscopy.

PubMed

Dukic, Maja; Todorov, Vencislav; Andany, Santiago; Nievergelt, Adrian P; Yang, Chen; Hosseini, Nahid; Fantner, Georg E

2017-12-01

Nearly all scanning probe microscopes (SPMs) contain a feedback controller, which is used to move the scanner in the direction of the z-axis in order to maintain a constant setpoint based on the tip-sample interaction. The most frequently used feedback controller in SPMs is the proportional-integral (PI) controller. The bandwidth of the PI controller presents one of the speed limiting factors in high-speed SPMs, where higher bandwidths enable faster scanning speeds and higher imaging resolution. Most SPM systems use digital signal processor-based PI feedback controllers, which require analog-to-digital and digital-to-analog converters. These converters introduce additional feedback delays which limit the achievable imaging speed and resolution. In this paper, we present a digitally controlled analog proportional-integral-derivative (PID) controller. The controller implementation allows tunability of the PID gains over a large amplification and frequency range, while also providing precise control of the system and reproducibility of the gain parameters. By using the analog PID controller, we were able to perform successful atomic force microscopy imaging of a standard silicon calibration grating at line rates up to several kHz.

A Phase Separation Route to Synthesize α-Fe2O3 Porous Nanofibers via Electrospinning for Ultrafast Ethanol Sensing

NASA Astrophysics Data System (ADS)

Dong, Shuwen; Yan, Shuang; Gao, Wenyuan; Liu, Guishan; Hao, Hongshun

2018-04-01

A facile and economic procedure was provided to synthesize α-Fe2O3 nanofibers. In this procedure, porous α-Fe2O3 nanofibers were obtained by a single-polymer/binary-solvent system, while solid α-Fe2O3 nanofibers were prepared by a single-polymer/single-solvent system. The crystal structure and morphology of both samples were characterized by x-ray diffraction, scanning electron microscopy, transmission electron microscopy and nitrogen adsorption/desorption isotherms. The formation mechanism of porous structure was based on solvent evaporation-induced phase separation by the use of mixed solvents with different volatility. Furthermore, ethanol-sensing performance of the porous α-Fe2O3 nanofibers was evaluated and compared with solid α-Fe2O3 nanofibers. Results from gas-sensing measurements reveal that porous α-Fe2O3 nanofibers exhibit higher sensitivity and slightly longer recovery time than solid α-Fe2O3 nanofibers. Over all, the gas sensor based on porous α-Fe2O3 nanofibers shows excellent ethanol-sensing capability with high sensitivity and ultrafast response/recovery behaviors, indicating its potential application as a real-time monitoring gas sensor.

Imaging of cellular spread on a three-dimensional scaffold by means of a novel cell-labeling technique for high-resolution computed tomography.

PubMed

Thimm, Benjamin W; Hofmann, Sandra; Schneider, Philipp; Carretta, Roberto; Müller, Ralph

2012-03-01

Computed tomography (CT) represents a truly three-dimensional (3D) imaging technique that can provide high-resolution images on the cellular level. Thus, one approach to detect single cells is X-ray absorption-based CT, where cells are labeled with a dense, opaque material providing the required contrast for CT imaging. Within the present work, a novel cell-labeling method has been developed showing the feasibility of labeling fixed cells with iron oxide (FeO) particles for subsequent CT imaging and quantitative morphometry. A biotin-streptavidin detection system was exploited to bind FeO particles to its target endothelial cells. The binding of the particles was predominantly close to the cell centers on 2D surfaces as shown by light microscopy, scanning electron microscopy, and CT. When cells were cultured on porous, 3D polyurethane surfaces, significantly more FeO particles were detected compared with surfaces without cells and FeO particle labeling using CT. Here, we report on the implementation and evaluation of a novel cell detection method based on high-resolution CT. This system has potential in cell tracking for 3D in vitro imaging in the future.

Gold nanoparticles-induced enhancement of the analytical response of an electrochemical biosensor based on an organic-inorganic hybrid composite material.

PubMed

Barbadillo, M; Casero, E; Petit-Domínguez, M D; Vázquez, L; Pariente, F; Lorenzo, E

2009-12-15

The design and characterization of a new organic-inorganic hybrid composite material for glucose electrochemical sensing are described. This material is based on the entrapment of both gold nanoparticles (AuNPs) and glucose oxidase, which was chosen as a model, into a sol-gel matrix. The addition of spectroscopic grade graphite to this system, which confers conductivity, leads to the development of a material particularly attractive for electrochemical biosensor fabrication. The characterization of the hybrid composite material was performed using atomic force microscopy and scanning electron microscopy techniques. This composite material was applied to the determination of glucose in presence of hydroxymethylferrocene as a redox mediator. The system exhibits a clear electrocatalytic activity towards glucose, allowing its determination at 250 mV vs Ag/AgCl. The performance of the resulting enzyme biosensor was evaluated in terms of sensitivity, detection limit, linear response range, stability and accuracy. Finally, the enhancement of the analytical response of the resulting biosensor induced by the presence of gold nanoparticles was evaluated by comparison with a similar organic-inorganic hybrid composite material without AuNPs.

Lightless cataract surgery using a near-infrared operating microscope.

PubMed

Kim, Bong-Hyun

2006-10-01

To describe the near-infrared (NIR) operating microscopy (NIOM) system using the NIR wavelength as the illumination source and to evaluate the feasibility of this system for lightless cataract surgery. HenAm Kim Eye Center, Haenam-Gun, South Korea. In this noncomparative interventional case series, cataract surgery was performed in 4 patients with bilateral cataract using the NIOM system in 1 eye and conventional microscopy in the fellow eye. The primary components of the system include an optical filter, a stereoscopic camera, head-mounted displays, and a recording system. This system uses invisible NIR (wavelength 850 to 1300 nm) illumination to facilitate cataract surgery without light. The differences between the NIOM system and conventional microscopy during cataract surgery were evaluated. The NIOM system provided excellent 3-dimensional viewing in real time. The image resolution was sufficient while performing all steps of cataract surgery. Immediately postoperatively and at 10 and 30 minutes and 1 hour, the visual acuity was better in the 4 eyes in which the NIOM system was used than in the 4 eyes in which conventional microscopy was used. However, using the NIOM system required good surgical skill. Lightless cataract surgery using the NIOM system seems useful for obtaining good visual acuity immediately postoperatively. The system may also reduce the incidence of light-induced retinal toxicity and the need for mydriatic administration and be a good educational tool.

Functionalization of a nanostructured hydroxyapatite with Cu(II) compounds as a pesticide: in situ transmission electron microscopy and environmental scanning electron microscopy observations of treated Vitis vinifera L. leaves.

PubMed

Battiston, Enrico; Salvatici, Maria C; Lavacchi, Alessandro; Gatti, Antonietta; Di Marco, Stefano; Mugnai, Laura

2018-02-19

The present study evaluated a biocompatible material for plant protection with the aim of reducing the amount of active substance applied. We used a synthetic hydroxyapatite (HA) that has been studied extensively as a consequence of its bioactivity and biocompatibility. An aggregation between HA nanoparticles and four Cu(II) compounds applied to Vitis vinifera L. leaves as a pesticide was studied. Formulations were characterized by X-ray diffraction (XRD), dynamic light scattering (DLS) and electron microscopy and applied in planta to verify particle aggregation and efficiency in controlling the pathogen Plasmopara viticola. The XRD patterns showed different crystalline phases dependig on the Cu(II) compound formulated with HA particles, DLS showed that nanostructured particles are stable as aggregates out of the nanometer range and, in all formulations, transmission electron microscopy (TEM) and environmental scanning electron microscopy (ESEM) microscopy showed large aggregates which were partially nanostructured and were recognized as stable in their micrometric dimensions. Such particles did not show phytotoxic effects after their application in planta. A formulation based on HA and a soluble Cu(II) compound showed promising results in the control of the fungal pathogen, confirming the potential role of HA as an innovative delivery system of Cu(II) ions. The present work indicates the possibility of improving the biological activity of a bioactive substance by modifying its structure through an achievable formulation with a biocompatible material. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy

NASA Astrophysics Data System (ADS)

Alfonso-García, Alba; Smith, Tim D.; Datta, Rupsa; Luu, Thuy U.; Gratton, Enrico; Potma, Eric O.; Liu, Wendy F.

2016-04-01

Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.

Autonomous Scanning Probe Microscopy in Situ Tip Conditioning through Machine Learning.

PubMed

Rashidi, Mohammad; Wolkow, Robert A

2018-05-23

Atomic-scale characterization and manipulation with scanning probe microscopy rely upon the use of an atomically sharp probe. Here we present automated methods based on machine learning to automatically detect and recondition the quality of the probe of a scanning tunneling microscope. As a model system, we employ these techniques on the technologically relevant hydrogen-terminated silicon surface, training the network to recognize abnormalities in the appearance of surface dangling bonds. Of the machine learning methods tested, a convolutional neural network yielded the greatest accuracy, achieving a positive identification of degraded tips in 97% of the test cases. By using multiple points of comparison and majority voting, the accuracy of the method is improved beyond 99%.

Ultrastructural observation of the acid-base resistant zone of all-in-one adhesives using three different acid-base challenges.

PubMed

Tsujimoto, Miho; Nikaido, Toru; Inoue, Go; Sadr, Alireza; Tagami, Junji

2010-11-01

The aim of this study was to analyze the ultrastructure of the dentin-adhesive interface using two all-in-one adhesive systems (Clearfil Tri-S Bond, TB; Tokuyama Bond Force, BF) after different acid-base challenges. Three solutions were used as acidic solutions for the acid-base challenges: a demineralizing solution (DS), a phosphoric acid solution (PA), and a hydrochloric acid solution (HCl). After the acid-base challenges, the bonded interfaces were examined by scanning electron microscopy. Thickness of the acid-base resistant zone (ABRZ) created in PA and HCl was thinner than in DS for both adhesive systems. For BF adhesive, an eroded area was observed beneath the ABRZ after immersion in PA and HCl, but not in DS. Conversely for TB adhesive, the eroded area was observed only after immersion in PA. In conclusion, although the ABRZ was observed for both all-in-one adhesive systems, its morphological features were influenced by the ingredients of both the adhesive material and acidic solution.

Application of a Loop-Type Laboratory Biofilm Reactor to the Evaluation of Biofilm for Some Metallic Materials and Polymers such as Urinary Stents and Catheters

PubMed Central

Kanematsu, Hideyuki; Kudara, Hikonaru; Kanesaki, Shun; Kogo, Takeshi; Ikegai, Hajime; Ogawa, Akiko; Hirai, Nobumitsu

2016-01-01

A laboratory biofilm reactor (LBR) was modified to a new loop-type closed system in order to evaluate novel stents and catheter materials using 3D optical microscopy and Raman spectroscopy. Two metallic specimens, pure nickel and cupronickel (80% Cu-20% Ni), along with two polymers, silicone and polyurethane, were chosen as examples to ratify the system. Each set of specimens was assigned to the LBR using either tap water or an NB (Nutrient broth based on peptone from animal foods and beef extract mainly)—cultured solution with E-coli formed over 48–72 h. The specimens were then analyzed using Raman Spectroscopy. 3D optical microscopy was employed to corroborate the Raman Spectroscopy results for only the metallic specimens since the inherent roughness of the polymer specimens made such measurements difficult. The findings suggest that the closed loop-type LBR together with Raman spectroscopy analysis is a useful method for evaluating biomaterials as a potential urinary system. PMID:28773945

In-situ straining and time-resolved electron tomography data acquisition in a transmission electron microscope.

PubMed

Hata, S; Miyazaki, S; Gondo, T; Kawamoto, K; Horii, N; Sato, K; Furukawa, H; Kudo, H; Miyazaki, H; Murayama, M

2017-04-01

This paper reports the preliminary results of a new in-situ three-dimensional (3D) imaging system for observing plastic deformation behavior in a transmission electron microscope (TEM) as a directly relevant development of the recently reported straining-and-tomography holder [Sato K et al. (2015) Development of a novel straining holder for transmission electron microscopy compatible with single tilt-axis electron tomography. Microsc. 64: 369-375]. We designed an integrated system using the holder and newly developed straining and image-acquisition software and then developed an experimental procedure for in-situ straining and time-resolved electron tomography (ET) data acquisition. The software for image acquisition and 3D visualization was developed based on the commercially available ET software TEMographyTM. We achieved time-resolved 3D visualization of nanometer-scale plastic deformation behavior in a Pb-Sn alloy sample, thus demonstrating the capability of this system for potential applications in materials science. © The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Nanogel-based natural polymers as smart carriers for the controlled delivery of Timolol Maleate through the cornea for glaucoma.

PubMed

Ilka, Roya; Mohseni, Mojdeh; Kianirad, Mehran; Naseripour, Masood; Ashtari, Khadijeh; Mehravi, Bita

2018-04-01

Despite frequent scientific efforts, efficient ocular drug delivery is a major challenge for pharmaceutical scientists. Poor bioavailability of ophthalmic solutions can be overcome by using smart ophthalmic drug-delivery systems. In this research, loading and delivery of Timolol Maleate (TM) through the cornea by synthesized nanoparticles based on biopolymers (chitosan-alginate) were studied. The physico-chemical properties of these nanoparticles were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and dynamic light scattering (DLS). Loading and release were evaluated by a UV-vis spectrometer and the ex vivo permeation study was carried out using the Franz Diffusion Cell and fluorescent microscopy studies. The results indicated that morphology and size of nanoparticles were spherical and in the range of 80-100nm. The loading capacity and encapsulation efficiency were about 42% and 94% respectively. They illustrated a burst release in the first hour followed by a slower and more sustained drug release during the next 24h. Also, the results indicated that the cornea penetration of TM loaded in nanoparticles was twice than that of TM. Hence, this nanocarrier can be considered as a suitable candidate for controlled TM delivery and release through the cornea. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of the five parameter grain boundary character distribution of nanocrystalline alpha-zirconium thin films using transmission electron microscopy

DOE PAGES

Ghamarian, I.; Samani, P.; Rohrer, G. S.; ...

2017-03-24

Grain boundary engineering and other fundamental materials science problems (e.g., phase transformations and physical properties) require an improvement in the understanding of the type and population of grain boundaries in a given system – yet, databases are limited in number and spare in detail, including for hcp crystals such as zirconium. One way to rapidly obtain databases to analyze is to use small-grained materials and high spatial resolution orientation microscopy techniques, such as ASTAR™/precession electron diffraction. To demonstrate this, a study of grain boundary character distributions was conducted for α-zirconium deposited at room temperature on fused silica substrates using physicalmore » vapor deposition. The orientation maps of the nanocrystalline thin films were acquired by the ASTARα/precession electron diffraction technique, a new transmission electron microscope based orientation microscopy method. The reconstructed grain boundaries were classified as pure tilt, pure twist, 180°-twist and 180°-tilt grain boundaries based on the distribution of grain boundary planes with respect to the angle/axis of misorientation associated with grain boundaries. The results of the current study were compared to the results of a similar study on α-titanium and the molecular dynamics results of grain boundary energy for α-titanium.« less

Monolithic Solid Based on Single-Walled Carbon Nanohorns: Preparation, Characterization, and Practical Evaluation as a Sorbent.

PubMed

Fresco-Cala, Beatriz; López-Lorente, Ángela I; Cárdenas, Soledad

2018-05-25

A monolithic solid based solely on single walled carbon nanohorns (SWNHs) was prepared without the need of radical initiators or gelators. The procedure involves the preparation of a wet jelly-like system of pristine SWNHs followed by slow drying (48 h) at 25 °C. As a result, a robust and stable porous network was formed due to the interaction between SWNHs not only via π-π and van der Waals interactions, but also via the formation of carbon bonds similar to those observed within dahlia aggregates. Pristine SWNHs and the SWNH monolith were characterized by several techniques, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), confocal laser scanning microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), and nitrogen intrusion porosimetry. Taking into account the efficiency of carbon nanoparticles in sorption processes, the potential applicability of the SWNH-monolith in this research field was explored using toluene; m-, p-, and o-xylene; ethylbenzene; and styrene, as target analytes. Detection limits were 0.01 µg·L -1 in all cases and the inter-day precision was in the interval 7.4⁻15.7%. The sorbent performance of the nanostructured monolithic solid was evaluated by extracting the selected compounds from different water samples with recovery values between 81.5% and 116.4%.

Deciphering complex, functional structures with synchrotron-based absorption and phase contrast tomographic microscopy

NASA Astrophysics Data System (ADS)

Stampanoni, M.; Reichold, J.; Weber, B.; Haberthür, D.; Schittny, J.; Eller, J.; Büchi, F. N.; Marone, F.

2010-09-01

Nowadays, thanks to the high brilliance available at modern, third generation synchrotron facilities and recent developments in detector technology, it is possible to record volumetric information at the micrometer scale within few minutes. High signal-to-noise ratio, quantitative information on very complex structures like the brain micro vessel architecture, lung airways or fuel cells can be obtained thanks to the combination of dedicated sample preparation protocols, in-situ acquisition schemes and cutting-edge imaging analysis instruments. In this work we report on recent experiments carried out at the TOMCAT beamline of the Swiss Light Source [1] where synchrotron-based tomographic microscopy has been successfully used to obtain fundamental information on preliminary models for cerebral fluid flow [2], to provide an accurate mesh for 3D finite-element simulation of the alveolar structure of the pulmonary acinus [3] and to investigate the complex functional mechanism of fuel cells [4]. Further, we introduce preliminary results on the combination of absorption and phase contrast microscopy for the visualization of high-Z nanoparticles in soft tissues, a fundamental information when designing modern drug delivery systems [5]. As an outlook we briefly discuss the new possibilities offered by high sensitivity, high resolution grating interferomtery as well as Zernike Phase contrast nanotomography [6].

Exploring lipids with nonlinear optical microscopy in multiple biological systems

NASA Astrophysics Data System (ADS)

Alfonso-Garcia, Alba

Lipids are crucial biomolecules for the well being of humans. Altered lipid metabolism may give rise to a variety of diseases that affect organs from the cardiovascular to the central nervous system. A deeper understanding of lipid metabolic processes would spur medical research towards developing precise diagnostic tools, treatment methods, and preventive strategies for reducing the impact of lipid diseases. Lipid visualization remains a complex task because of the perturbative effect exerted by traditional biochemical assays and most fluorescence markers. Coherent Raman scattering (CRS) microscopy enables interrogation of biological samples with minimum disturbance, and is particularly well suited for label-free visualization of lipids, providing chemical specificity without compromising on spatial resolution. Hyperspectral imaging yields large datasets that benefit from tailored multivariate analysis. In this thesis, CRS microscopy was combined with Raman spectroscopy and other label-free nonlinear optical techniques to analyze lipid metabolism in multiple biological systems. We used nonlinear Raman techniques to characterize Meibum secretions in the progression of dry eye disease, where the lipid and protein contributions change in ratio and phase segregation. We employed similar tools to examine lipid droplets in mice livers aboard a spaceflight mission, which lose their retinol content contributing to the onset of nonalcoholic fatty-liver disease. We also focused on atherosclerosis, a disease that revolves around lipid-rich plaques in arterial walls. We examined the lipid content of macrophages, whose variable phenotype gives rise to contrasting healing and inflammatory activities. We also proposed new label-free markers, based on lifetime imaging, for macrophage phenotype, and to detect products of lipid oxidation. Cholesterol was also detected in hepatitis C virus infected cells, and in specific strains of age-related macular degeneration diseased cells by spontaneous Raman spectroscopy. We used synthesized highly-deuterated cholesterol to track its compartmentalization in adrenal cells, revealing heterogeneous lipid droplet content. These examples illustrate the potential of label-free nonlinear optical microscopy for unveiling complex physiological processes by direct visualization of lipids. Detailed image analysis and combined microscopy modalities will continue to reveal and quantify fundamental biology that will support the advance of biomedicine.

Evaluation of mobile digital light-emitting diode fluorescence microscopy in Hanoi, Viet Nam.

PubMed

Chaisson, L H; Reber, C; Phan, H; Switz, N; Nilsson, L M; Myers, F; Nhung, N V; Luu, L; Pham, T; Vu, C; Nguyen, H; Nguyen, A; Dinh, T; Nahid, P; Fletcher, D A; Cattamanchi, A

2015-09-01

Hanoi Lung Hospital, Hanoi, Viet Nam. To compare the accuracy of CellScopeTB, a manually operated mobile digital fluorescence microscope, with conventional microscopy techniques. Patients referred for sputum smear microscopy to the Hanoi Lung Hospital from May to September 2013 were included. Ziehl-Neelsen (ZN) smear microscopy, conventional light-emitting diode (LED) fluorescence microscopy (FM), CellScopeTB-based LED FM and Xpert(®) MTB/RIF were performed on sputum samples. The sensitivity and specificity of microscopy techniques were determined in reference to Xpert results, and differences were compared using McNemar's paired test of proportions. Of 326 patients enrolled, 93 (28.5%) were Xpert-positive for TB. The sensitivity of ZN microscopy, conventional LED FM, and CellScopeTB-based LED FM was respectively 37.6% (95%CI 27.8-48.3), 41.9% (95%CI 31.8-52.6), and 35.5% (95%CI 25.8-46.1). The sensitivity of CellScopeTB was similar to that of conventional LED FM (difference -6.5%, 95%CI -18.2 to 5.3, P = 0.33) and ZN microscopy (difference -2.2%, 95%CI -9.2 to 4.9, P = 0.73). The specificity was >99% for all three techniques. CellScopeTB performed similarly to conventional microscopy techniques in the hands of experienced TB microscopists. However, the sensitivity of all sputum microscopy techniques was low. Options enabled by digital microscopy, such as automated imaging with real-time computerized analysis, should be explored to increase sensitivity.

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2013-01-01

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

The OptIPuter microscopy demonstrator: enabling science through a transatlantic lightpath

PubMed Central

Ellisman, M.; Hutton, T.; Kirkland, A.; Lin, A.; Lin, C.; Molina, T.; Peltier, S.; Singh, R.; Tang, K.; Trefethen, A.E.; Wallom, D.C.H.; Xiong, X.

2009-01-01

The OptIPuter microscopy demonstrator project has been designed to enable concurrent and remote usage of world-class electron microscopes located in Oxford and San Diego. The project has constructed a network consisting of microscopes and computational and data resources that are all connected by a dedicated network infrastructure using the UK Lightpath and US Starlight systems. Key science drivers include examples from both materials and biological science. The resulting system is now a permanent link between the Oxford and San Diego microscopy centres. This will form the basis of further projects between the sites and expansion of the types of systems that can be remotely controlled, including optical, as well as electron, microscopy. Other improvements will include the updating of the Microsoft cluster software to the high performance computing (HPC) server 2008, which includes the HPC basic profile implementation that will enable the development of interoperable clients. PMID:19487201

The OptIPuter microscopy demonstrator: enabling science through a transatlantic lightpath.

PubMed

Ellisman, M; Hutton, T; Kirkland, A; Lin, A; Lin, C; Molina, T; Peltier, S; Singh, R; Tang, K; Trefethen, A E; Wallom, D C H; Xiong, X

2009-07-13

The OptIPuter microscopy demonstrator project has been designed to enable concurrent and remote usage of world-class electron microscopes located in Oxford and San Diego. The project has constructed a network consisting of microscopes and computational and data resources that are all connected by a dedicated network infrastructure using the UK Lightpath and US Starlight systems. Key science drivers include examples from both materials and biological science. The resulting system is now a permanent link between the Oxford and San Diego microscopy centres. This will form the basis of further projects between the sites and expansion of the types of systems that can be remotely controlled, including optical, as well as electron, microscopy. Other improvements will include the updating of the Microsoft cluster software to the high performance computing (HPC) server 2008, which includes the HPC basic profile implementation that will enable the development of interoperable clients.

Determination of 200 °C Isothermal Section of Al-Ag-Ga Phase Diagram by Microanalysis, X-ray Diffraction, Hardness and Electrical Conductivity Measurements

NASA Astrophysics Data System (ADS)

Premović, Milena; Tomović, Milica; Minić, Duško; Manasijević, Dragan; Živković, Dragana; Ćosović, Vladan; Grković, Vladan; Đorđević, Aleksandar

2017-04-01

Ternary Al-Ag-Ga system at 200 °C was experimentally and thermodynamically assessed. Isothermal section was extrapolated using optimized thermodynamic parameters for constitutive binary systems. Microstructure and phase composition of the selected alloy samples were analyzed using light microscopy, scanning electron microscopy combined with energy-dispersive spectrometry and x-ray powder diffraction technique. The obtained experimental results were found to be in a close agreement with the predicted phase equilibria. Hardness and electrical conductivity of the alloy samples from four vertical sections Al-Ag80Ga20, Al-Ag60Ga40, Ag-Al80Ga20 and Ag-Al60Ga40 of the ternary Al-Ag-Ga system at 200 °C were experimentally determined using Brinell method and eddy current measurements. Additionally, hardness of the individual phases present in the microstructure of the studied alloy samples was determined using Vickers microhardness test. Based on experimentally obtained results, isolines of Brinell hardness and electrical conductivity were calculated for the alloys from isothermal section of the ternary Al-Ag-Ga system at 200 °C.

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.

Microcontroller-driven fluid-injection system for atomic force microscopy.

PubMed

Kasas, S; Alonso, L; Jacquet, P; Adamcik, J; Haeberli, C; Dietler, G

2010-01-01

We present a programmable microcontroller-driven injection system for the exchange of imaging medium during atomic force microscopy. Using this low-noise system, high-resolution imaging can be performed during this process of injection without disturbance. This latter circumstance was exemplified by the online imaging of conformational changes in DNA molecules during the injection of anticancer drug into the fluid chamber.

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.

Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution.

PubMed

Yamashiro, Sawako; Watanabe, Naoki

2017-07-06

Live-cell single-molecule imaging was introduced more than a decade ago, and has provided critical information on remodeling of the actin cytoskeleton, the motion of plasma membrane proteins, and dynamics of molecular motor proteins. Actin remodeling has been the best target for this approach because actin and its associated proteins stop diffusing when assembled, allowing visualization of single-molecules of fluorescently-labeled proteins in a state specific manner. The approach based on this simple principle is called Single-Molecule Speckle (SiMS) microscopy. For instance, spatiotemporal regulation of actin polymerization and lifetime distribution of actin filaments can be monitored directly by tracking actin SiMS. In combination with fluorescently labeled probes of various actin regulators, SiMS microscopy has contributed to clarifying the processes underlying recycling, motion and remodeling of the live-cell actin network. Recently, we introduced an electroporation-based method called eSiMS microscopy, with high efficiency, easiness and improved spatiotemporal precision. In this review, we describe the application of live-cell single-molecule imaging to cellular actin dynamics and discuss the advantages of eSiMS microscopy over previous SiMS microscopy.

Quantification of surface displacements and electromechanical phenomena via dynamic atomic force microscopy

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

Balke, Nina; Jesse, Stephen; Yu, Pu

Detection of dynamic surface displacements associated with local changes in material strain provides access to a number of phenomena and material properties. Contact resonance-enhanced methods of atomic force microscopy (AFM) have been shown capable of detecting ~1–3 pm-level surface displacements, an approach used in techniques such as piezoresponse force microscopy, atomic force acoustic microscopy, and ultrasonic force microscopy. Here, based on an analytical model of AFM cantilever vibrations, we demonstrate a guideline to quantify surface displacements with high accuracy by taking into account the cantilever shape at the first resonant contact mode, depending on the tip–sample contact stiffness. The approachmore » has been experimentally verified and further developed for piezoresponse force microscopy (PFM) using well-defined ferroelectric materials. These results open up a way to accurate and precise measurements of surface displacement as well as piezoelectric constants at the pm-scale with nanometer spatial resolution and will allow avoiding erroneous data interpretations and measurement artifacts. Furthermore, this analysis is directly applicable to all cantilever-resonance-based scanning probe microscopy (SPM) techniques.« less

Quantification of surface displacements and electromechanical phenomena via dynamic atomic force microscopy

DOE PAGES

Balke, Nina; Jesse, Stephen; Yu, Pu; ...

2016-09-15

Detection of dynamic surface displacements associated with local changes in material strain provides access to a number of phenomena and material properties. Contact resonance-enhanced methods of atomic force microscopy (AFM) have been shown capable of detecting ~1–3 pm-level surface displacements, an approach used in techniques such as piezoresponse force microscopy, atomic force acoustic microscopy, and ultrasonic force microscopy. Here, based on an analytical model of AFM cantilever vibrations, we demonstrate a guideline to quantify surface displacements with high accuracy by taking into account the cantilever shape at the first resonant contact mode, depending on the tip–sample contact stiffness. The approachmore » has been experimentally verified and further developed for piezoresponse force microscopy (PFM) using well-defined ferroelectric materials. These results open up a way to accurate and precise measurements of surface displacement as well as piezoelectric constants at the pm-scale with nanometer spatial resolution and will allow avoiding erroneous data interpretations and measurement artifacts. Furthermore, this analysis is directly applicable to all cantilever-resonance-based scanning probe microscopy (SPM) techniques.« less

X-ray microscopy using reflection targets based on SEM with tungsten filament

NASA Astrophysics Data System (ADS)

Liu, Junbiao; Ma, Yutian; Zhao, Weixia; Niu, Geng; Chu, Mingzhang; Yin, Bohua; Han, Li; Liu, Baodong

2016-10-01

X-ray MicroandNano imaging is developed based on the conventional x-ray tomography, it can not only provide nondestructive testing with higher resolution measurement, but also be used to examine the material or the structure with low atomic number and low density. The source with micro-focal spot size is one of the key components of x-ray MicroandNano imaging. The focused electron beam from SEM bombarding the metal target can generate x-ray with ultra-small size. It is convenient to set up x-ray microscopy based on SEM for laboratory use. This paper describes a new x-ray microscopy using reflection targets based on FEI Quanta600 SEM with tungsten filament. The flat panel detector is placed outside of the vacuum chamber with 300μm thickness Be-window to isolate vacuum from the air. A stage with 3 DOFs is added to adjust the positions of the target, the SEM's sample stage is used to move sample. And the shape of target is designed as cone with 60° half cone angle to get the maximum x-ray dosage. The attenuation coefficient of Bewindow for x-ray is about 25%. Finally, the line pair card is used to evaluate the resolution and the result shows that the resolution of the system can receive less than 750nm, when the acceleration voltage is 30keV, the beam current is 160nA, the SEM working distance is 5mm and the acquisition time of the detector is 60s.

Measurement of the refractive index of solutions based on digital holographic microscopy

NASA Astrophysics Data System (ADS)

Huang, Sujuan; Wang, Weiping; Zeng, Junzhang; Yan, Cheng; Lin, Yunyi; Wang, Tingyun

2018-01-01

A new approach for the refractive index (RI) measurement of solutions is proposed based on digital holographic microscopy. The experimental system consists of a modified Mach-Zehnder interferometer and related lab-developed analysis software. The high quality digital hologram of the tested solution is obtained by the real-time analysis software, which is firstly encapsulated into a capillary tube, and then the capillary tube is inserted in a matching fluid. An angular spectrum algorithm is adopted to extract the phase distribution from the hologram recorded by a CCD. Based on a capillary multi-layer calculation model, the RI of the tested solution is obtained at high accuracy. The results of transparent glycerol solution measured by the proposed method are more accurate than those measured by the Abbe refractometer. We also measure the RI of translucent magnetic fluid, which is not suitable to be measured by the Abbe refractometer. The relationship between the RI and the concentration of magnetic fluid is experimentally studied, and the results show that the RI is linearly related to the concentration of dilute magnetic fluid.

Automated analysis of siRNA screens of cells infected by hepatitis C and dengue viruses based on immunofluorescence microscopy images

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

2008-03-01

We present an image analysis approach as part of a high-throughput microscopy siRNA-based screening system using cell arrays for the identification of cellular genes involved in hepatitis C and dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in the neighborhood of segmented cell nuclei, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment and single images. In particular, we propose a novel approach for the localization of regions of transfected cells within cell array images, which combines model-based circle fitting and grid fitting. By this scheme we integrate information from single cell array images and knowledge from the complete cell arrays. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behaviour of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

Hyperspectral Systems Increase Imaging Capabilities

NASA Technical Reports Server (NTRS)

2010-01-01

In 1983, NASA started developing hyperspectral systems to image in the ultraviolet and infrared wavelengths. In 2001, the first on-orbit hyperspectral imager, Hyperion, was launched aboard the Earth Observing-1 spacecraft. Based on the hyperspectral imaging sensors used in Earth observation satellites, Stennis Space Center engineers and Institute for Technology Development researchers collaborated on a new design that was smaller and used an improved scanner. Featured in Spinoff 2007, the technology is now exclusively licensed by Themis Vision Systems LLC, of Richmond, Virginia, and is widely used in medical and life sciences, defense and security, forensics, 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.

Genetically encoded sensors and fluorescence microscopy for anticancer research

NASA Astrophysics Data System (ADS)

Zagaynova, Elena V.; Shirmanova, Marina V.; Sergeeva, Tatiana F.; Klementieva, Natalia V.; Mishin, Alexander S.; Gavrina, Alena I.; Zlobovskay, Olga A.; Furman, Olga E.; Dudenkova, Varvara V.; Perelman, Gregory S.; Lukina, Maria M.; Lukyanov, Konstantin A.

2017-02-01

Early response of cancer cells to chemical compounds and chemotherapeutic drugs were studied using novel fluorescence tools and microscopy techniques. We applied confocal microscopy, two-photon fluorescence lifetime imaging microscopy and super-resolution localization-based microscopy to assess structural and functional changes in cancer cells in vitro. The dynamics of energy metabolism, intracellular pH, caspase-3 activation during staurosporine-induced apoptosis as well as actin cytoskeleton rearrangements under chemotherapy were evaluated. We have showed that new genetically encoded sensors and advanced fluorescence microscopy methods provide an efficient way for multiparameter analysis of cell activities

Scanning probe recognition microscopy investigation of tissue scaffold properties

PubMed Central

Fan, Yuan; Chen, Qian; Ayres, Virginia M; Baczewski, Andrew D; Udpa, Lalita; Kumar, Shiva

2007-01-01

Scanning probe recognition microscopy is a new scanning probe microscopy technique which enables selective scanning along individual nanofibers within a tissue scaffold. Statistically significant data for multiple properties can be collected by repetitively fine-scanning an identical region of interest. The results of a scanning probe recognition microscopy investigation of the surface roughness and elasticity of a series of tissue scaffolds are presented. Deconvolution and statistical methods were developed and used for data accuracy along curved nanofiber surfaces. Nanofiber features were also independently analyzed using transmission electron microscopy, with results that supported the scanning probe recognition microscopy-based analysis. PMID:18203431

Scanning probe recognition microscopy investigation of tissue scaffold properties.

PubMed

Fan, Yuan; Chen, Qian; Ayres, Virginia M; Baczewski, Andrew D; Udpa, Lalita; Kumar, Shiva

2007-01-01

Scanning probe recognition microscopy is a new scanning probe microscopy technique which enables selective scanning along individual nanofibers within a tissue scaffold. Statistically significant data for multiple properties can be collected by repetitively fine-scanning an identical region of interest. The results of a scanning probe recognition microscopy investigation of the surface roughness and elasticity of a series of tissue scaffolds are presented. Deconvolution and statistical methods were developed and used for data accuracy along curved nanofiber surfaces. Nanofiber features were also independently analyzed using transmission electron microscopy, with results that supported the scanning probe recognition microscopy-based analysis.

Combined two-photon microscopy and optical coherence tomography using individually optimized sources

NASA Astrophysics Data System (ADS)

Jeong, Bosu; Lee, Byunghak; Jang, Min Seong; Nam, Hyoseok; Kim, Hae Koo; Yoon, Sang June; Doh, Junsang; Lee, Sang-Joon; Yang, Bo-Gie; Jang, Myoung Ho; Kim, Ki Hean

2011-03-01

Two-photon microscopy (TPM) and optical coherence tomography (OCT) are 3D tissue imaging techniques based on different contrast mechanisms. We developed a combined system of TPM and OCT to provide information of both imaging modalities for in-vivo tissue study. TPM and OCT were implemented by using separate light sources, a Ti-Sapphire laser and a wavelength-swept source centered at 1300 nm respectively, and scanners. Light from the two sources was combined for the simultaneous imaging of tissue samples. TPM provided molecular, cellular information of tissues in the region of a few hundred microns on one side at a sub-cellular resolution, and ran at approximately 40 frames per second. OCT provided structural information in the tissue region larger than TPM images at a sub-tenth micron resolution by using 0.1 numerical aperture. OCT had the field of view of 800 um × 800 um based on a 20x objective, the sensitivity of 97dB, and the imaging speed of 0.8 volumes per second. This combined system was tested with simple microsphere specimens, and then was applied to image the explanted intestine of a mouse model and the plant leaves. Morphology and micro-structures of the intestine villi and immune cells within the villi were shown in the intestine image, and chloroplasts and various microstructures of the maize leaves were visualized in 3D by the combined system.

Microfluidic device for a rapid immobilization of zebrafish larvae in environmental scanning electron microscopy.

PubMed

Akagi, Jin; Zhu, Feng; Skommer, Joanna; Hall, Chris J; Crosier, Philip S; Cialkowski, Michal; Wlodkowic, Donald

2015-03-01

Small vertebrate model organisms have recently gained popularity as attractive experimental models that enhance our understanding of human tissue and organ development. Despite a large body of evidence using optical spectroscopy for the characterization of small model organism on chip-based devices, no attempts have been so far made to interface microfabricated technologies with environmental scanning electron microscopy (ESEM). Conventional scanning electron microscopy requires high vacuum environments and biological samples must be, therefore, submitted to many preparative procedures to dehydrate, fix, and subsequently stain the sample with gold-palladium deposition. This process is inherently low-throughput and can introduce many analytical artifacts. This work describes a proof-of-concept microfluidic chip-based system for immobilizing zebrafish larvae for ESEM imaging that is performed in a gaseous atmosphere, under low vacuum mode and without any need for sample staining protocols. The microfabricated technology provides a user-friendly and simple interface to perform ESEM imaging on zebrafish larvae. Presented lab-on-a-chip device was fabricated using a high-speed infrared laser micromachining in a biocompatible poly(methyl methacrylate) thermoplastic. It consisted of a reservoir with multiple semispherical microwells designed to hold the yolk of dechorionated zebrafish larvae. Immobilization of the larvae was achieved by a gentle suction generated during blotting of the medium. Trapping region allowed for multiple specimens to be conveniently positioned on the chip-based device within few minutes for ESEM imaging. © 2014 International Society for Advancement of Cytometry.

Are rapid diagnostic tests more accurate in diagnosis of plasmodium falciparum malaria compared to microscopy at rural health centres?

PubMed

Batwala, Vincent; Magnussen, Pascal; Nuwaha, Fred

2010-12-02

Prompt, accurate diagnosis and treatment with artemisinin combination therapy remains vital to current malaria control. Blood film microscopy the current standard test for diagnosis of malaria has several limitations that necessitate field evaluation of alternative diagnostic methods especially in low income countries of sub-Saharan Africa where malaria is endemic. The accuracy of axillary temperature, health centre (HC) microscopy, expert microscopy and a HRP2-based rapid diagnostic test (Paracheck) was compared in predicting malaria infection using polymerase chain reaction (PCR) as the gold standard. Three hundred patients with a clinical suspicion of malaria based on fever and or history of fever from a low and high transmission setting in Uganda were consecutively enrolled and provided blood samples for all tests. Accuracy of each test was calculated overall with 95% confidence interval and then adjusted for age-groups and level of transmission intensity using a stratified analysis. The endpoints were: sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). This study is registered with Clinicaltrials.gov, NCT00565071. Of the 300 patients, 88(29.3%) had fever, 56(18.7%) were positive by HC microscopy, 47(15.7%) by expert microscopy, 110(36.7%) by Paracheck and 89(29.7%) by PCR. The overall sensitivity >90% was only shown by Paracheck 91.0% [95%CI: 83.1-96.0]. The sensitivity of expert microscopy was 46%, similar to HC microscopy. The superior sensitivity of Paracheck compared to microscopy was maintained when data was stratified for transmission intensity and age. The overall specificity rates were: Paracheck 86.3% [95%CI: 80.9-90.6], HC microscopy 93.4% [95%CI: 89.1-96.3] and expert microscopy 97.2% [95%CI: 93.9-98.9]. The NPV >90% was shown by Paracheck 95.8% [95%CI: 91.9-98.2]. The overall PPV was <88% for all methods. The HRP2-based RDT has shown superior sensitivity compared to microscopy in diagnosis of malaria and may be more suitable for screening of malaria infection.

Identifying and overcoming the interface originating c-axis instability in highly Sc enhanced AlN for piezoelectric micro-electromechanical systems

NASA Astrophysics Data System (ADS)

Fichtner, Simon; Wolff, Niklas; Krishnamurthy, Gnanavel; Petraru, Adrian; Bohse, Sascha; Lofink, Fabian; Chemnitz, Steffen; Kohlstedt, Hermann; Kienle, Lorenz; Wagner, Bernhard

2017-07-01

Enhancing the piezoelectric activity of AlN by partially substituting Al with Sc to form Al1-xScxN is a promising approach to improve the performance of piezoelectric micro-electromechanical systems. Here, we present evidence of an instability in the morphology of Al1-xScxN, which originates at, or close to, the substrate/Al1-xScxN interface and becomes more pronounced as the Sc content is increased. Based on Transmission electron microscopy, piezoresponse force microscopy, X-ray diffraction, and SEM analysis, it is identified to be the incipient formation of (100) oriented grains. Approaches to successfully reestablish exclusive c-axis orientation up to x = 0.43 are revealed, with electrode pre-treatment and cathode-substrate distance found to exert significant influence. This allows us to present first measurements of the transversal thin film piezoelectric coefficient e31,f and dielectric loss tangent tan δ beyond x = 0.3.

Portable microscopy platform for the clinical and environmental monitoring

NASA Astrophysics Data System (ADS)

Wang, Weiming; Yu, Yan; Huang, Hui; Ou, Jinping

2016-04-01

Light microscopy can not only address various diagnosis needs such as aquatic parasites and bacteria such as E. coli in water, but also provide a method for the screening of red tide. Traditional microscope based on the smartphone created by adding lens couldn't keep the tradeoff between field-of-view(FOV) and the resolution. In this paper, we demonstrate a non-contact, light and cost-effective microscope platform, that can image highly dense samples with a spatial resolution of ~0.8um over a field-of-view(FOV) of >1mm2. After captured the direct images, we performed the pixel super-resolution algorithm to improve the image resolution and overcome the hardware interference. The system would be a good point-of-care diagnostic solution in resource limited settings. We validated the performance of the system by imaging resolution test targets, the squamous cell cancer(SqCC) and green algae that necessary to detect the squamous carcinoma and red tide

Effects of spatial coherence in diffraction phase microscopy.

PubMed

Edwards, Chris; Bhaduri, Basanta; Nguyen, Tan; Griffin, Benjamin G; Pham, Hoa; Kim, Taewoo; Popescu, Gabriel; Goddard, Lynford L

2014-03-10

Quantitative phase imaging systems using white light illumination can exhibit lower noise figures than laser-based systems. However, they can also suffer from object-dependent artifacts, such as halos, which prevent accurate reconstruction of the surface topography. In this work, we show that white light diffraction phase microscopy using a standard halogen lamp can produce accurate height maps of even the most challenging structures provided that there is proper spatial filtering at: 1) the condenser to ensure adequate spatial coherence and 2) the output Fourier plane to produce a uniform reference beam. We explain that these object-dependent artifacts are a high-pass filtering phenomenon, establish design guidelines to reduce the artifacts, and then apply these guidelines to eliminate the halo effect. Since a spatially incoherent source requires significant spatial filtering, the irradiance is lower and proportionally longer exposure times are needed. To circumvent this tradeoff, we demonstrate that a supercontinuum laser, due to its high radiance, can provide accurate measurements with reduced exposure times, allowing for fast dynamic measurements.

Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy.

PubMed

Melo, Rossana C N; Weller, Peter F

2016-10-01

Electron microscopy (EM)-based techniques are mostly responsible for our current view of cell morphology at the subcellular level and continue to play an essential role in biological research. In cells from the immune system, such as eosinophils, EM has helped to understand how cells package and release mediators involved in immune responses. Ultrastructural investigations of human eosinophils enabled visualization of secretory processes in detail and identification of a robust, vesicular trafficking essential for the secretion of immune mediators via a non-classical secretory pathway associated with secretory (specific) granules. This vesicular system is mainly organized as large tubular-vesicular carriers (Eosinophil Sombrero Vesicles - EoSVs) actively formed in response to cell activation and provides a sophisticated structural mechanism for delivery of granule-stored mediators. In this review, we highlight the application of EM techniques to recognize pools of immune mediators at vesicular compartments and to understand the complex secretory pathway within human eosinophils involved in inflammatory and allergic responses. Copyright © 2016 Elsevier Inc. All rights reserved.

CognitionMaster: an object-based image analysis framework

PubMed Central

2013-01-01

Background Automated image analysis methods are becoming more and more important to extract and quantify image features in microscopy-based biomedical studies and several commercial or open-source tools are available. However, most of the approaches rely on pixel-wise operations, a concept that has limitations when high-level object features and relationships between objects are studied and if user-interactivity on the object-level is desired. Results In this paper we present an open-source software that facilitates the analysis of content features and object relationships by using objects as basic processing unit instead of individual pixels. Our approach enables also users without programming knowledge to compose “analysis pipelines“ that exploit the object-level approach. We demonstrate the design and use of example pipelines for the immunohistochemistry-based cell proliferation quantification in breast cancer and two-photon fluorescence microscopy data about bone-osteoclast interaction, which underline the advantages of the object-based concept. Conclusions We introduce an open source software system that offers object-based image analysis. The object-based concept allows for a straight-forward development of object-related interactive or fully automated image analysis solutions. The presented software may therefore serve as a basis for various applications in the field of digital image analysis. PMID:23445542

Procedures for analysis of debris relative to Space Shuttle systems

NASA Technical Reports Server (NTRS)

Kim, Hae Soo; Cummings, Virginia J.

1993-01-01

Debris samples collected from various Space Shuttle systems have been submitted to the Microchemical Analysis Branch. This investigation was initiated to develop optimal techniques for the analysis of debris. Optical microscopy provides information about the morphology and size of crystallites, particle sizes, amorphous phases, glass phases, and poorly crystallized materials. Scanning electron microscopy with energy dispersive spectrometry is utilized for information on surface morphology and qualitative elemental content of debris. Analytical electron microscopy with wavelength dispersive spectrometry provides information on the quantitative elemental content of debris.

Towards a quantitative description of tunneling conductance of superconductors: Application to LiFeAs

DOE PAGES

Kreisel, A.; Nelson, R.; Berlijn, T.; ...

2016-12-27

Since the discovery of iron-based superconductors, a number of theories have been put forward to explain the qualitative origin of pairing, but there have been few attempts to make quantitative, material-specific comparisons to experimental results. The spin-fluctuation theory of electronic pairing, based on first-principles electronic structure calculations, makes predictions for the superconducting gap. Within the same framework, the surface wave functions may also be calculated, allowing, e.g., for detailed comparisons between theoretical results and measured scanning tunneling topographs and spectra. We present such a comparison between theory and experiment on the Fe-based superconductor LiFeAs. Our results for the homogeneous surfacemore » as well as impurity states are presented as a benchmark test of the theory. For the homogeneous system, we argue that the maxima of topographic image intensity may be located at positions above either the As or Li atoms, depending on tip height and the setpoint current of the measurement. We further report the experimental observation of transitions between As- and Li-registered lattices as functions of both tip height and setpoint bias, in agreement with this prediction. Next, we give a detailed comparison between the simulated scanning tunneling microscopy images of transition-metal defects with experiment. Finally, we discuss possible extensions of the current framework to obtain a theory with true predictive power for scanning tunneling microscopy in Fe-based systems.« less

Towards a quantitative description of tunneling conductance of superconductors: Application to LiFeAs

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

Kreisel, A.; Nelson, R.; Berlijn, T.

Since the discovery of iron-based superconductors, a number of theories have been put forward to explain the qualitative origin of pairing, but there have been few attempts to make quantitative, material-specific comparisons to experimental results. The spin-fluctuation theory of electronic pairing, based on first-principles electronic structure calculations, makes predictions for the superconducting gap. Within the same framework, the surface wave functions may also be calculated, allowing, e.g., for detailed comparisons between theoretical results and measured scanning tunneling topographs and spectra. We present such a comparison between theory and experiment on the Fe-based superconductor LiFeAs. Our results for the homogeneous surfacemore » as well as impurity states are presented as a benchmark test of the theory. For the homogeneous system, we argue that the maxima of topographic image intensity may be located at positions above either the As or Li atoms, depending on tip height and the setpoint current of the measurement. We further report the experimental observation of transitions between As- and Li-registered lattices as functions of both tip height and setpoint bias, in agreement with this prediction. Next, we give a detailed comparison between the simulated scanning tunneling microscopy images of transition-metal defects with experiment. Finally, we discuss possible extensions of the current framework to obtain a theory with true predictive power for scanning tunneling microscopy in Fe-based systems.« less

Systematic, spatial imaging of large multimolecular assemblies and the emerging principles of supramolecular order in biological systems

PubMed Central

Schubert, Walter

2013-01-01

Understanding biological systems at the level of their relational (emergent) molecular properties in functional protein networks relies on imaging methods, able to spatially resolve a tissue or a cell as a giant, non-random, topologically defined collection of interacting supermolecules executing myriads of subcellular mechanisms. Here, the development and findings of parameter-unlimited functional super-resolution microscopy are described—a technology based on the fluorescence imaging cycler (IC) principle capable of co-mapping thousands of distinct biomolecular assemblies at high spatial resolution and differentiation (<40 nm distances). It is shown that the subcellular and transcellular features of such supermolecules can be described at the compositional and constitutional levels; that the spatial connection, relational stoichiometry, and topology of supermolecules generate hitherto unrecognized functional self-segmentation of biological tissues; that hierarchical features, common to thousands of simultaneously imaged supermolecules, can be identified; and how the resulting supramolecular order relates to spatial coding of cellular functionalities in biological systems. A large body of observations with IC molecular systems microscopy collected over 20 years have disclosed principles governed by a law of supramolecular segregation of cellular functionalities. This pervades phenomena, such as exceptional orderliness, functional selectivity, combinatorial and spatial periodicity, and hierarchical organization of large molecular systems, across all species investigated so far. This insight is based on the high degree of specificity, selectivity, and sensitivity of molecular recognition processes for fluorescence imaging beyond the spectral resolution limit, using probe libraries controlled by ICs. © 2013 The Authors. Journal of Molecular Recognition published by John Wiley & Sons, Ltd. PMID:24375580

Formation of Worm-Like Micelles in Mixed N-Hexadecyl-N-Methylpyrrolidinium Bromide-Based Cationic Surfactant and Anionic Surfactant Systems

PubMed Central

Dai, Caili; Yan, Zhihu; You, Qing; Du, Mingyong; Zhao, Mingwei

2014-01-01

Through the descriptive and rheological characterization of worm-like micelles formed by N-hexadecyl-N-methylpyrrolidinium bromide and sodium laurate, the formation and properties of the worm-like micelles were affected by the concentrations of sodium laurate and temperature. Additionally, cryogenic transmission electron microscopy images further validated the formation of worm-like micelles. PMID:25019152

Optical sectioning using a digital Fresnel incoherent-holography-based confocal imaging system

PubMed Central

Kelner, Roy; Katz, Barak; Rosen, Joseph

2015-01-01

We propose a new type of confocal microscope using Fresnel incoherent correlation holography (FINCH). Presented here is a confocal configuration of FINCH using a phase pinhole and point illumination that is able to suppress out-of-focus information from the recorded hologram and hence combine the super-resolution capabilities of FINCH with the sectioning capabilities of confocal microscopy. PMID:26413560

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 role of cell body density in ruminant retina mechanics assessed by atomic force and Brillouin microscopy

NASA Astrophysics Data System (ADS)

Weber, Isabell P.; Yun, Seok Hyun; Scarcelli, Giuliano; Franze, Kristian

2017-12-01

Cells in the central nervous system (CNS) respond to the stiffness of their environment. CNS tissue is mechanically highly heterogeneous, thus providing motile cells with region-specific mechanical signals. While CNS mechanics has been measured with a variety of techniques, reported values of tissue stiffness vary greatly, and the morphological structures underlying spatial changes in tissue stiffness remain poorly understood. We here exploited two complementary techniques, contact-based atomic force microscopy and contact-free Brillouin microscopy, to determine the mechanical properties of ruminant retinae, which are built up by different tissue layers. As in all vertebrate retinae, layers of high cell body densities (‘nuclear layers’) alternate with layers of low cell body densities (‘plexiform layers’). Different tissue layers varied significantly in their mechanical properties, with the photoreceptor layer being the stiffest region of the retina, and the inner plexiform layer belonging to the softest regions. As both techniques yielded similar results, our measurements allowed us to calibrate the Brillouin microscopy measurements and convert the Brillouin shift into a quantitative assessment of elastic tissue stiffness with optical resolution. Similar as in the mouse spinal cord and the developing Xenopus brain, we found a strong correlation between nuclear densities and tissue stiffness. Hence, the cellular composition of retinae appears to strongly contribute to local tissue stiffness, and Brillouin microscopy shows a great potential for the application in vivo to measure the mechanical properties of transparent tissues.

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

PubMed Central

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

2008-01-01

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

Development and Application of Chemical Probes for Vibrational Imaging by Stimulated Raman Scattering

NASA Astrophysics Data System (ADS)

Hu, Fanghao

During the last decade, Raman microscopy is experiencing rapid development and increasingly applied in biological and medical systems. Especially, stimulated Raman scattering (SRS) microscopy, which significantly improves the sensitivity of Raman scattering through stimulated emission, has allowed direct visualization of many species that are previously challenging with conventional fluorescence imaging. Compared to fluorescence, SRS imaging requires no label or small label on the target molecule, thus with minimal perturbation to the molecule of interest. Moreover, Raman scattering is free from complicated photophysical and photochemical processes such as photobleaching, and has intrinsically narrower linewidth than fluorescence emission. This allows multiplexed Raman imaging with minimal spectral crosstalk and excellent photo-stability. To achieve the full potential of Raman microscopy, vibrational probes have been developed for Raman imaging. Multiple Raman probes with a few atoms in size are applied in Raman imaging with high sensitivity and specificity. An overview of both fluorescence and Raman microscopy and their imaging probes is given in Chapter 1 with a brief discussion on the SRS theory. Built on the current progress of Raman microscopy and vibrational probes, I write on my research in the development of carbon-deuterium, alkyne and nitrile probes for visualizing choline metabolism (Chapter 2), glucose uptake activity (Chapter 3), complex brain metabolism (Chapter 4) and polymeric nanoparticles (Chapter 5) in live cells and tissues, as well as the development of polyyne-based vibrational probes for super-multiplexed imaging, barcoding and analysis (Chapter 6).

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

PubMed

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

2008-07-16

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

Feasibility and diagnostic accuracy of Internet-based dynamic telepathology between Uganda and Germany.

PubMed

Wamala, Dan; Katamba, Achilles; Dworak, Otto

2011-01-01

We assessed the feasibility and diagnostic accuracy of Internet-based telepathology compared with conventional microscopic examination. A total of 96 cases from the routine workload of the Department of Pathology at the Mulago Hospital in Uganda were examined by robotic telemicroscopy via the Internet at the Fuerth Hospital in Germany. The telepathology diagnoses were compared with those of conventional microscopy. Email and Skype telephony were used to exchange clinical and diagnostic information. The reference diagnosis (gold standard) was established by consensus between two or more experienced pathologists using both conventional microscopy and telemicroscopy; immunohistochemistry was used whenever it was necessary. It took approximately 30 min for a pathologist to learn to use the telepathology system and 4-25 min to read a case remotely. Internet speed was the main limiting factor. The images were of good quality and the pathologist at the remote site was able to navigate through the slide and change the magnification as necessary. In 92 of the specimens (97%), the pathologists at the two hospitals agreed exactly about the diagnosis. Agreement overall was moderate (kappa = 0.39). The discordant diagnoses were attributed to factors related to diseases morphologically difficult to diagnose, such as soft tissue sarcomas and primitive tumours. Internet-based conferencing systems offer an inexpensive method of obtaining a primary diagnosis by telepathology and consulting on cases that require subspecialty expertise.

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.

Portable lensless wide-field microscopy imaging platform based on digital inline holography and multi-frame pixel super-resolution

PubMed Central

Sobieranski, Antonio C; Inci, Fatih; Tekin, H Cumhur; Yuksekkaya, Mehmet; Comunello, Eros; Cobra, Daniel; von Wangenheim, Aldo; Demirci, Utkan

2017-01-01

In this paper, an irregular displacement-based lensless wide-field microscopy imaging platform is presented by combining digital in-line holography and computational pixel super-resolution using multi-frame processing. The samples are illuminated by a nearly coherent illumination system, where the hologram shadows are projected into a complementary metal-oxide semiconductor-based imaging sensor. To increase the resolution, a multi-frame pixel resolution approach is employed to produce a single holographic image from multiple frame observations of the scene, with small planar displacements. Displacements are resolved by a hybrid approach: (i) alignment of the LR images by a fast feature-based registration method, and (ii) fine adjustment of the sub-pixel information using a continuous optimization approach designed to find the global optimum solution. Numerical method for phase-retrieval is applied to decode the signal and reconstruct the morphological details of the analyzed sample. The presented approach was evaluated with various biological samples including sperm and platelets, whose dimensions are in the order of a few microns. The obtained results demonstrate a spatial resolution of 1.55 µm on a field-of-view of ≈30 mm2. PMID:29657866

Real-time quantum cascade laser-based infrared microspectroscopy in-vivo

NASA Astrophysics Data System (ADS)

Kröger-Lui, N.; Haase, K.; Pucci, A.; Schönhals, A.; Petrich, W.

2016-03-01

Infrared microscopy can be performed to observe dynamic processes on a microscopic scale. Fourier-transform infrared spectroscopy-based microscopes are bound to limitations regarding time resolution, which hampers their potential for imaging fast moving systems. In this manuscript we present a quantum cascade laser-based infrared microscope which overcomes these limitations and readily achieves standard video frame rates. The capabilities of our setup are demonstrated by observing dynamical processes at their specific time scales: fermentation, slow moving Amoeba Proteus and fast moving Caenorhabditis elegans. Mid-infrared sampling rates between 30 min and 20 ms are demonstrated.

Label-free imaging of cellular malformation using high resolution photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Chen, Zhongjiang; Li, Bingbing; Yang, Sihua

2014-09-01

A label-free high resolution photoacoustic microscopy (PAM) system for imaging cellular malformation is presented. The carbon fibers were used to testify the lateral resolution of the PAM. Currently, the lateral resolution is better than 2.7 μm. The human normal red blood cells (RBCs) were used to prove the imaging capability of the system, and a single red blood cell was mapped with high contrast. Moreover, the iron deficiency anemia RBCs were clearly distinguished from the cell morphology by using the PAM. The experimental results demonstrate that the photoacoustic microscopy system can accomplish label-free photoacoustic imaging and that it has clinical potential for use in the detection of erythrocytes and blood vessels malformation.

A multiplexed chip-based assay system for investigating the functional development of human skeletal myotubes in vitro.

PubMed

Smith, A S T; Long, C J; Pirozzi, K; Najjar, S; McAleer, C; Vandenburgh, H H; Hickman, J J

2014-09-20

This report details the development of a non-invasive in vitro assay system for investigating the functional maturation and performance of human skeletal myotubes. Data is presented demonstrating the survival and differentiation of human myotubes on microscale silicon cantilevers in a defined, serum-free system. These cultures can be stimulated electrically and the resulting contraction quantified using modified atomic force microscopy technology. This system provides a higher degree of sensitivity for investigating contractile waveforms than video-based analysis, and represents the first system capable of measuring the contractile activity of individual human muscle myotubes in a reliable, high-throughput and non-invasive manner. The development of such a technique is critical for the advancement of body-on-a-chip platforms toward application in pre-clinical drug development screens. Copyright © 2014 Elsevier B.V. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2016-07-01

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

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

PubMed Central

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

2016-01-01

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

3D reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout

PubMed Central

Hodgkinson, Julie L.; Horsley, Ashley; Stabat, David; Simon, Martha; Johnson, Steven; da Fonseca, Paula C. A.; Morris, Edward P.; Wall, Joseph S.; Lea, Susan M.; Blocker, Ariel J.

2009-01-01

Type III secretion systems (T3SSs) mediate bacterial protein translocation into eukaryotic cells, a process essential for virulence of many Gram-negative pathogens. They are composed of a cytoplasmic secretion machinery and a base bridging both bacterial membranes into which a hollow, external needle is embedded. When isolated, the latter two parts are termed ‘needle complex’ (NC). Incomplete understanding of NC structure hampers studies of T3SS function. To estimate the stoichiometry of its components, the mass f its sub-domains was measured by scanning transmission electron microscopy (STEM). Subunit symmetries were determined by analysis of top and side views within negatively stained samples in low dose transmission electron microscopy (TEM). Application of 12-fold symmetry allowed generation of a 21-25Å resolution three-dimensional (3D) reconstruction of the NC base, revealing many new features and permitting tentative docking of the crystal structure of EscJ, an inner membrane component. PMID:19396171

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

NASA Astrophysics Data System (ADS)

Ryu, Inkeon; Kim, Daekeun

2018-04-01

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

Molecular engineering of two-photon fluorescent probes for bioimaging applications

NASA Astrophysics Data System (ADS)

Liu, Hong-Wen; Liu, Yongchao; Wang, Peng; Zhang, Xiao-Bing

2017-03-01

During the past two decades, two-photon microscopy (TPM), which utilizes two near-infrared photons as the excitation source, has emerged as a novel, attractive imaging tool for biological research. Compared with one-photon microscopy, TPM offers several advantages, such as lowering background fluorescence in living cells and tissues, reducing photodamage to biosamples, and a photobleaching phenomenon, offering better 3D spatial localization, and increasing penetration depth. Small-molecule-based two-photon fluorescent probes have been well developed for the detection and imaging of various analytes in biological systems. In this review, we will give a general introduction of molecular engineering of two-photon fluorescent probes based on different fluorescence response mechanisms for bioimaging applications during the past decade. Inspired by the desired advantages of small-molecule two-photon fluorescent probes in biological imaging applications, we expect that more attention will be devoted to the development of new two-photon fluorophores and applications of TPM in areas of bioanalysis and disease diagnosis.

Strain-dependent characterization of electrode and polymer network of electrically activated polymer actuators

NASA Astrophysics Data System (ADS)

Töpper, Tino; Osmani, Bekim; Weiss, Florian M.; Winterhalter, Carla; Wohlfender, Fabian; Leung, Vanessa; Müller, Bert

2015-04-01

Fecal incontinence describes the involuntary loss of bowel content and affects about 45 % of retirement home residents and overall more than 12 % of the adult population. Artificial sphincter implants for treating incontinence are currently based on mechanical systems with failure rates resulting in revision after three to five years. To overcome this drawback, artificial muscle sphincters based on bio-mimetic electro-active polymer (EAP) actuators are under development. Such implants require polymer films that are nanometer-thin, allowing actuation below 24 V, and electrodes that are stretchable, remaining conductive at strains of about 10 %. Strain-dependent resistivity measurements reveal an enhanced conductivity of 10 nm compared to 30 nm sputtered Au on silicone for strains higher than 5 %. Thus, strain-dependent morphology characterization with optical microscopy and atomic force microscopy could demonstrate these phenomena. Cantilever bending measurements are utilized to determine elastic/viscoelastic properties of the EAP films as well as their long-term actuation behavior. Controlling these properties enables the adjustment of growth parameters of nanometer-thin EAP actuators.

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.

Cooperative light-induced molecular movements of highly ordered azobenzene self-assembled monolayers.

PubMed

Pace, Giuseppina; Ferri, Violetta; Grave, Christian; Elbing, Mark; von Hänisch, Carsten; Zharnikov, Michael; Mayor, Marcel; Rampi, Maria Anita; Samorì, Paolo

2007-06-12

Photochromic systems can convert light energy into mechanical energy, thus they can be used as building blocks for the fabrication of prototypes of molecular devices that are based on the photomechanical effect. Hitherto a controlled photochromic switch on surfaces has been achieved either on isolated chromophores or within assemblies of randomly arranged molecules. Here we show by scanning tunneling microscopy imaging the photochemical switching of a new terminally thiolated azobiphenyl rigid rod molecule. Interestingly, the switching of entire molecular 2D crystalline domains is observed, which is ruled by the interactions between nearest neighbors. This observation of azobenzene-based systems displaying collective switching might be of interest for applications in high-density data storage.

Corrosion Resistance of a Cast-Iron Material Coated With a Ceramic Layer Using Thermal Spray Method

NASA Astrophysics Data System (ADS)

Florea, C. D.; Bejinariu, C.; Munteanu, C.; Istrate, B.; Toma, S. L.; Alexandru, A.; Cimpoesu, R.

2018-06-01

Cast-iron 250 used for breake systems present many corrosion signs after a mean usage time based on the environment conditions they work. In order to improve them corrosion resistance we propose to cover the active part of the material using a ceramic material. The deposition process is an industrial deposition system based on thermal spraying that can cover high surfaces in low time. In this articol we analyze the influence of a ceramic layer (40-50 µm) on the corrosion resistance of FC250 cast iron. The results were analyzed using scanning electron microscopy (SEM), X-ray energy dispersive (EDS) and linear and cyclic potentiometry.

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

PubMed

Kubota, Yoshiyuki

2015-02-01

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

Automated Dermoscopy Image Analysis of Pigmented Skin Lesions

PubMed Central

Baldi, Alfonso; Quartulli, Marco; Murace, Raffaele; Dragonetti, Emanuele; Manganaro, Mario; Guerra, Oscar; Bizzi, Stefano

2010-01-01

Dermoscopy (dermatoscopy, epiluminescence microscopy) is a non-invasive diagnostic technique for the in vivo observation of pigmented skin lesions (PSLs), allowing a better visualization of surface and subsurface structures (from the epidermis to the papillary dermis). This diagnostic tool permits the recognition of morphologic structures not visible by the naked eye, thus opening a new dimension in the analysis of the clinical morphologic features of PSLs. In order to reduce the learning-curve of non-expert clinicians and to mitigate problems inherent in the reliability and reproducibility of the diagnostic criteria used in pattern analysis, several indicative methods based on diagnostic algorithms have been introduced in the last few years. Recently, numerous systems designed to provide computer-aided analysis of digital images obtained by dermoscopy have been reported in the literature. The goal of this article is to review these systems, focusing on the most recent approaches based on content-based image retrieval systems (CBIR). PMID:24281070

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.

Effect of the calcium silicate-based sealer removal protocols and time-point of acid etching on the dentin adhesive interface.

PubMed

Morais, Jéssika Mayhara Pereira; Victorino, Keli Regina; Escalante-Otárola, Wilfredo Gustavo; Jordão-Basso, Keren Cristina Fagundes; Palma-Dibb, Regina Guenka; Kuga, Milton Carlos

2018-06-15

The aim of the study was to evaluate the effects when acid etching on the dentin surface was immediately performed (I) or 7 days (D) after calcium silicate-based sealer (MTA Fillapex) removal, using 95% ethanol (E) or xylol (X). First study, 60 bovine incisor dentin specimens were impregnated with sealer and divided into six groups (n = 10): (EI), E + I; (XI), X + I; (ED), E + D; (XD), X + D, (UN), untreated and (MR), mechanical removal of sealer. Scanning electron microscopy (SEM) images (500×) were obtained from each specimen and scores assessed the sealer residues persistence. Second study, 60 specimens were similarly treated; however, the specimens were restored with composite resin after the removal protocols. Hybrid layer formation was evaluated using confocal laser microscopy (1,024×). Third study, 60 specimens were similarly obtained and subjected to micro-shear test to evaluate the effects of removal protocols on the bond strength of etch-and- rinse adhesive system to dentin. XI showed the highest persistence of sealer residues (p < .05), similar to MR (p > .05). EI showed the greatest hybrid layer extension, except in relation to UN (p < .05). XI and MR presented the lowest bond strength adhesive system to dentin (p < .05). Acid etching immediately after calcium silicate-based endodontic sealer removal using xylol presented the highest residues persistence and negatively affected the adhesive interface between dentin and etch-and-rinse adhesive system. © 2018 Wiley Periodicals, Inc.

Direct Writing of Graphene-based Nanoelectronics via Atomic Force Microscopy

DTIC Science & Technology

2012-05-07

To) 07-05-2012 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Direct Writing of Graphene -based Nanoelectronics via Atomic Force Microscopy 5b. GRANT...ABSTRACT This project employs direct writing with an atomic force microscope (AFM) to fabricate simple graphene -based electronic components like resistors...and transistors at nanometer-length scales. The goal is to explore their electrical properties for graphene -based electronics. Conducting

Recommendations for the design and the installation of large laser scanning microscopy systems

NASA Astrophysics Data System (ADS)

Helm, P. Johannes

2012-03-01

Laser Scanning Microscopy (LSM) has since the inventions of the Confocal Scanning Laser Microscope (CLSM) and the Multi Photon Laser Scanning Microscope (MPLSM) developed into an essential tool in contemporary life science and material science. The market provides an increasing number of turn-key and hands-off commercial LSM systems, un-problematic to purchase, set up and integrate even into minor research groups. However, the successful definition, financing, acquisition, installation and effective use of one or more large laser scanning microscopy systems, possibly of core facility character, often requires major efforts by senior staff members of large academic or industrial units. Here, a set of recommendations is presented, which are helpful during the process of establishing large systems for confocal or non-linear laser scanning microscopy as an effective operational resource in the scientific or industrial production process. Besides the description of technical difficulties and possible pitfalls, the article also illuminates some seemingly "less scientific" processes, i.e. the definition of specific laboratory demands, advertisement of the intention to purchase one or more large systems, evaluation of quotations, establishment of contracts and preparation of the local environment and laboratory infrastructure.

Identification and restoration in 3D fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dieterlen, Alain; Xu, Chengqi; Haeberle, Olivier; Hueber, Nicolas; Malfara, R.; Colicchio, B.; Jacquey, Serge

2004-06-01

3-D optical fluorescent microscopy becomes now an efficient tool for volumic investigation of living biological samples. The 3-D data can be acquired by Optical Sectioning Microscopy which is performed by axial stepping of the object versus the objective. For any instrument, each recorded image can be described by a convolution equation between the original object and the Point Spread Function (PSF) of the acquisition system. To assess performance and ensure the data reproducibility, as for any 3-D quantitative analysis, the system indentification is mandatory. The PSF explains the properties of the image acquisition system; it can be computed or acquired experimentally. Statistical tools and Zernike moments are shown appropriate and complementary to describe a 3-D system PSF and to quantify the variation of the PSF as function of the optical parameters. Some critical experimental parameters can be identified with these tools. This is helpful for biologist to define an aquisition protocol optimizing the use of the system. Reduction of out-of-focus light is the task of 3-D microscopy; it is carried out computationally by deconvolution process. Pre-filtering the images improves the stability of deconvolution results, now less dependent on the regularization parameter; this helps the biologists to use restoration process.

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

Leung, Bonnie; Hitchcock, Adam; Brash, John

Spun-cast films of polystyrene (PS) blended with polylactide (PLA) were visualized and characterized using atomic force microscopy (AFM) and synchrotron-based X-ray photoemission electron microscopy (X-PEEM). The composition of the two polymers in these systems was determined by quantitative chemical analysis of near-edge X-ray absorption signals recorded with X-PEEM. The surface morphology depends on the ratio of the two components, the total polymer concentration, and the temperature of vacuum annealing. For most of the blends examined, PS is the continuous phase with PLA existing in discrete domains or segregated to the air?polymer interface. Phase segregation was improved with further annealing. Amore » phase inversion occurred when films of a 40:60 PS:PLA blend (0.7 wt percent loading) were annealed above the glass transition temperature (Tg) of PLA.« less

Laser-assisted formation of micropores and nanobubbles in sclera promote stable normalization of intraocular pressure

NASA Astrophysics Data System (ADS)

Baum, Olga; Wachsmann-Hogiu, Sebastian; Milner, Thomas; Sobol, Emil

2017-06-01

Pores in sclera enhance uveoscleral water outflow and can normalize intraocular pressure in glaucomatous eyes. The aims of this study are to demonstrate laser-induced formation of pores with a dendritic structure and to answer the questions: How is a pore system stable and can laser treatment provide a long-lasting pressure stabilization effect? Effect of 1.56 µm laser radiation on porcine eye sclera was studied using atomic force microscopy and super resolution structured irradiation microscopy with fluorescent markers. Results suggest that the pores with a complex spatial configuration can arise as a result of laser irradiation and that laser-generated stable gas nanobubbles coated with calcium ions allow pore stabilization in the sclera. Our results support a laser based approach for treatment of glaucoma.

Analysis of beat fluctuations and oxygen consumption in cardiomyocytes by scanning electrochemical microscopy.

PubMed

Hirano, Yu; Kodama, Mikie; Shibuya, Masahiro; Maki, Yoshiyuki; Komatsu, Yasuo

2014-02-15

The contractile behavior of cardiomyocytes can be monitored by measuring their action potentials, and the analysis is essential for screening the safety of potential drugs. However, immobilizing cardiac cells on a specific electrode is considerably complicated. In this study, we demonstrate that scanning electrochemical microscopy (SECM) can be used to analyze rapid topographic changes in beating cardiomyocytes in a standard culture dish. Various cardiomyocyte contraction parameters and oxygen consumption based on cell respiration could be determined from SECM data. We also confirmed that cellular changes induced by adding the cardiotonic agent digoxin were conveniently monitored by this SECM system. These results show that SECM can be a potentially powerful tool for use in drug development for cardiovascular diseases. Copyright © 2013 Elsevier Inc. All rights reserved.

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

Patel, Sajan; Petty, Clayton W.; Krafcik, Karen Lee

Electrostatic modes of atomic force microscopy have shown to be non-destructive and relatively simple methods for imaging conductors embedded in insulating polymers. Here we use electrostatic force microscopy to image the dispersion of carbon nanotubes in a latex-based conductive composite, which brings forth features not observed in previously studied systems employing linear polymer films. A fixed-potential model of the probe-nanotube electrostatics is presented which in principle gives access to the conductive nanoparticle's depth and radius, and the polymer film dielectric constant. Comparing this model to the data results in nanotube depths that appear to be slightly above the film–air interface.more » Furthermore, this result suggests that water-mediated charge build-up at the film–air interface may be the source of electrostatic phase contrast in ambient conditions.« less

Cryo diffraction microscopy: Ice conditions and finite supports

DOE PAGES

Miao, H.; Downing, K.; Huang, X.; ...

2009-09-25

Using a signal-to-noise ratio estimation based on correlations between multiple simulated images, we compare the dose efficiency of two soft x-ray imaging systems: incoherent brightfield imaging using zone plate optics in a transmission x-ray microscope (TXM), and x-ray diffraction microscopy (XDM) where an image is reconstructed from the far-field coherent diffraction pattern. In XDM one must computationally phase weak diffraction signals; in TXM one suffers signal losses due to the finite numerical aperture and efficiency of the optics. In simulations with objects representing isolated cells such as yeast, we find that XDM has the potential for delivering equivalent resolution imagesmore » using fewer photons. This can be an important advantage for studying radiation-sensitive biological and soft matter specimens.« less

Ballistic-Electron-Emission-Microscopy of Strained Si(sub 1-x)Ge(sub x) Layers

NASA Technical Reports Server (NTRS)

Bell, L. D.; Milliken, A. M.; Manion, S. J.; Kaiser, W. J.; Fathauer, R. W.; Pike, W. T.

1994-01-01

Ballistic-electron-emission microscopy (BEEM) has been used to investigate the effects of strain on Si(sub 1-x)Ge(sub x) alloys. Lifting of the degeneracy of the conduction-band minimum of Si(sub 1-x)Ge(sub x), due to lattice deformation has been directly measured by application of BEEM spectroscopy to Ag/Si structures. Experimental values for this conduction-band splitting agree well with calculations. In addition, an unexpected heterogeneity in the strain of the Si(sub 1-x)Ge(sub x) layer is introduced by deposition of Au. This effect, not observed with Ag, is attributed to species interdiffusion and has important implications for metal-semiconductor devices based oil pseudomorphic Si(sub 1-x)Ge(sub x)/Si material systems.

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

PubMed

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

2016-12-01

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

Applications of AFM for atomic manipulation and spectroscopy

NASA Astrophysics Data System (ADS)

Custance, Oscar

2009-03-01

Since the first demonstration of atom-by-atom assembly [1], atomic manipulation with scanning tunneling microscopy has yielded stunning realizations in nanoscience. A new exciting panorama has been recently opened with the possibility of manipulating atoms at surfaces using atomic force microscopy (AFM) [2-5]. In this talk, we will present two different approaches that enable patterning structures at semiconductor surfaces by manipulating individual atoms with AFM and at room temperature [2, 3]. We will discuss the physics behind each protocol through the analysis of the measured forces associated with these manipulations [3-5]. Another challenging issue in scanning probe microscopy is the ability to disclose the local chemical composition of a multi-element system at atomic level. Here, we will introduce a single-atom chemical identification method, which is based on detecting the forces between the outermost atom of the AFM tip and the atoms at a surface [6]. We demonstrate this identification procedure on a particularly challenging system, where any discrimination attempt based solely on topographic measurements would be impossible to achieve. [4pt] References: [0pt] [1] D. M. Eigler and E. K. Schweizer, Nature 344, 524 (1990); [0pt] [2] Y. Sugimoto, M. Abe, S. Hirayama, N. Oyabu, O. Custance and S. Morita, Nature Materials 4, 156 (2005); [0pt] [3] Y. Sugimoto, P. Pou, O. Custance, P. Jelinek, M. Abe, R. Perez and S. Morita, Science 322, 413 (2008); [0pt] [4] Y. Sugimoto, P. Jelinek, P. Pou, M. Abe, S. Morita, R. Perez and O. Custance, Phys. Rev. Lett. 98, 106104 (2007); [0pt] [5] M. Ternes, C. P. Lutz, C. F. Hirjibehedin, F. J. Giessibl and A. J. Heinrich, Science 319, 1066 (2008); [0pt] [6] Y. Sugimoto, P. Pou, M. Abe, P. Jelinek, R. Perez, S. Morita, and O. Custance, Nature 446, 64 (2007)

Platinum replica electron microscopy: Imaging the cytoskeleton globally and locally.

PubMed

Svitkina, Tatyana M

2017-05-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the "comfort zones" of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platinum Replica Electron Microscopy: Imaging the Cytoskeleton Globally and Locally

PubMed Central

SVITKINA, Tatyana M.

2017-01-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the “comfort zones” of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. PMID:28323208

Synthesis of MAX Phases in the Hf-Al-C System.

PubMed

Lapauw, Thomas; Tunca, Bensu; Cabioc'h, Thierry; Lu, Jun; Persson, Per O Å; Lambrinou, Konstantina; Vleugels, Jozef

2016-11-07

For the first time, MAX phases in the Hf-Al-C system were experimentally synthesized using reactive hot pressing. HfC was observed as the main competing phase. The lattice parameters of Hf 2 AlC and Hf 3 AlC 2 were determined by Rietveld refinement based on the X-ray diffraction data. The atomic stacking sequence was revealed by high-resolution scanning transmission electron microscopy. Mixtures of 211 and 312 stacking were observed within the same grain, including 523 layers. This transition in atomic structure is discussed.

[Organization of olfactory system of the Indian major carp Labeo rohita (Ham.): a study using scanning and transmission microscopy].

PubMed

Bhute, Y V; Baile, V V

2007-01-01

Catla catla, Labeo rohita, and Cirrhinus mrigala are important alimentary fish in India. Their reproduction (breeding) depends on season. The fish perceive external factors-stimuli and chemical signals through the olfactory system that plays the key role in the central regulation of reproduction. However, in the available literature, any electron microscopy data on organization of olfactory elements in these fish are absent. We have studied ultrastructure of the olfactory organ in male L. rohita by using scanning (SEM) and transmission electron microscopy (TEM). The olfactory organ consists of olfactory epithelium, a short nerve, and olfactory bulb. The organ has oval shape and consists of approximately 47-52 lamellae in adult fish and of 14-20 lamellae in fish at the stage of fingerling. These lamellae originate from the midline raphe. By using SEM, the presence of microvillar sensory and ciliated non-sensory cells in these lamellae is shown. By using TEM, a microvillar receptor cell is revealed, which has rough endoplasmic reticulum and Golgi apparatus towards the apical end. Basal cells are found at the base of the receptor cell; supporting cells are located adjacent to olfactory receptor neurons, while epithelial cells--in the non-sensory part of olfactory epithelium. Mast, blastema and macrophages cells are also found in the basal lamina. This work is the first publication on structural organization of olfactory system of the Indian major carp, which provides information about morphological and ultrastructural organization of olfactory system and opens new opportunities for study of chemical neuroanatomy, sensory signal processing, and nervous regulation of reproduction of the Indian major carp.

Silicon nanowires reliability and robustness investigation using AFM-based techniques

NASA Astrophysics Data System (ADS)

Bieniek, Tomasz; Janczyk, Grzegorz; Janus, Paweł; Grabiec, Piotr; Nieprzecki, Marek; Wielgoszewski, Grzegorz; Moczała, Magdalena; Gotszalk, Teodor; Buitrago, Elizabeth; Badia, Montserrat F.; Ionescu, Adrian M.

2013-07-01

Silicon nanowires (SiNWs) have undergone intensive research for their application in novel integrated systems such as field effect transistor (FET) biosensors and mass sensing resonators profiting from large surface-to-volume ratios (nano dimensions). Such devices have been shown to have the potential for outstanding performances in terms of high sensitivity, selectivity through surface modification and unprecedented structural characteristics. This paper presents the results of mechanical characterization done for various types of suspended SiNWs arranged in a 3D array. The characterization has been performed using techniques based on atomic force microscopy (AFM). This investigation is a necessary prerequisite for the reliable and robust design of any biosensing system. This paper also describes the applied investigation methodology and reports measurement results aggregated during series of AFM-based tests.

NRC/AMRMC Resident Research Associateship Program

DTIC Science & Technology

2013-03-01

l) Intravital Microscopy was successfully employed for investigating EG sheddÍng in hemorrhagic shock/resuscitation for the very first time; 2... Intravital microscopy integrated with systemic hemodynamics evaluations may be essential and more accurate tools to identify changes and study mechanisms...Glycocalyx during Hemorrhagic Shock in Rats using Intravital Microscopy. Submitted to Microvascular Research, 2012 (under review). FINAL REPORT Laboratory

NRC/AMRMC Resident Research Associateship Program

DTIC Science & Technology

2013-04-01

Intravital Microscopy was successfully employed for investigating EG shedding in hemorrhagic shock/resuscitation for the very first time; Contract... Intravital microscopy integrated with systemic hemodynamics evaluations may be essential and more accurate tools to identify changes and study...findings in concise form, utilizing key concepts/words. 1) Intravital Microscopy was successfully employed for investigating EG shedding in hemorrhagic

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

PubMed Central

Wolenski, Joseph S.; Julich, Doerthe

2014-01-01

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy. PMID:24600334

Fluorescence Microscopy of Single Molecules

ERIC Educational Resources Information Center

Zimmermann, Jan; van Dorp, Arthur; Renn, Alois

2004-01-01

The investigation of photochemistry and photophysics of individual quantum systems is described with the help of a wide-field fluorescence microscopy approach. The fluorescence single molecules are observed in real time.

In vivo magnetic resonance microscopy of brain structure in unanesthetized flies

NASA Astrophysics Data System (ADS)

Jasanoff, Alan; Sun, Phillip Z.

2002-09-01

We present near-cellular-resolution magnetic resonance (MR) images of an unanesthetized animal, the blowfly Sarcophaga bullata. Immobilized flies were inserted into a home-built gradient probe in a 14.1-T magnet, and images of voxel size (20-40 μm) 3—comparable to the diameter of many neuronal cell bodies in the fly's brain—were obtained in several hours. Use of applied field gradients on the order of 60 G/cm allowed minimally distorted images to be produced, despite significant susceptibility differences across the specimen. The images we obtained have exceptional contrast-to-noise levels; comparison with histology-based anatomical information shows that the MR microscopy faithfully represents patterns of nervous tissue and allows distinct brain regions to be clearly identified. Even at the highest resolutions we explored, morphological detail was pronounced in the apparent absence of instabilities or movement-related artifacts frequently observed during imaging of live animal specimens. This work demonstrates that the challenges of noninvasive in vivo MR microscopy can be overcome in a system amenable to studies of brain structure and physiology.

Self-assembled monolayers of alkyl-thiols on InAs: A Kelvin probe force microscopy study

NASA Astrophysics Data System (ADS)

Szwajca, A.; Wei, J.; Schukfeh, M. I.; Tornow, M.

2015-03-01

We report on the preparation and characterization of self-assembled monolayers from aliphatic thiols with different chain length and termination on InAs (100) planar surfaces. This included as first step the development and investigation of a thorough chemical InAs surface preparation step using a dedicated bromine/NH4OH-based etching process. Ellipsometry, contact angle measurements and atomic force microscopy (AFM) indicated the formation of smooth, surface conforming monolayers. The molecular tilt angles were obtained as 30 ± 10° with respect to the surface normal. Kelvin probe force microscopy (KPFM) measurements in hand with Parameterized Model number 5 (PM5) calculations of the involved molecular dipoles allowed for an estimation of the molecular packing densities on the surface. We obtained values of up to n = 1014 cm- 2 for the SAMs under study. These are close to what is predicted from a simple geometrical model that would calculate a maximum density of about n = 2.7 × 1014 cm- 2. We take this as additional conformation of the substrate smoothness and quality of our InAs-SAM hybrid layer systems.

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

PubMed

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

2012-07-01

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

Intravital phosphorescence lifetime imaging of the renal cortex accurately measures renal hypoxia.

PubMed

Hirakawa, Yosuke; Mizukami, Kiichi; Yoshihara, Toshitada; Takahashi, Ippei; Khulan, Purevsuren; Honda, Tomoko; Mimura, Imari; Tanaka, Tetsuhiro; Tobita, Seiji; Nangaku, Masaomi

2018-06-01

Renal tubulointerstitial hypoxia is recognized as a final common pathway of chronic kidney disease and is considered a promising drug target. However, hypoxia in the tubules is not well examined because of limited detection methods. Here, we devised a method to visualize renal tubular oxygen tension with spatial resolution at a cellular level using the cell-penetrating phosphorescent probe, BTPDM1 (an iridium-based cationic lipophilic dye), and confocal phosphorescence lifetime imaging microscopy to precisely assess renal hypoxia. Imaging with BTPDM1 revealed an oxygen gradient between S1 and S2 segments in mouse kidney. We also demonstrated that our microscopy system can detect subtle changes of hypoxemia and reoxygenation, and the acquired phosphorescence lifetime can be converted to partial pressure of oxygen. This new method allows, for the first time, visualization of intravital oxygen gradients at the renal surface with high spatial resolution. Thus, the confocal phosphorescence lifetime imaging microscopy platform, combined with BTPDM1, will promote an accurate understanding of tissue hypoxia, including renal hypoxia. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Triticella minini - a new ctenostome bryozoan from the abyssal plain adjacent to the Kuril-Kamchatka Trench

NASA Astrophysics Data System (ADS)

Grischenko, Andrei V.; Chernyshev, Alexei V.

2015-01-01

A new species of ctenostome bryozoan, Triticella minini sp. nov., is described from the abyssal plain adjacent to the Kuril-Kamchatka Trench, based on material collected by the Russian-German deep-sea expedition KuramBio 2012. Colonies of T. minini sp. nov. were found attached to the oral spines of irregular sea urchin Echinosigra (Echinogutta) amphoraMironov, 1974 by means of rhizoid fibers that penetrated the substratum through circular borings. The specimens were examined by light microscopy, scanning electron microscopy, and confocal laser scanning microscopy with phalloidin and nuclear labeling. The description of T. minini sp. nov. combines a general taxonomic description with a description of the anatomy of the muscular system. The new species differs from congeners in lacking a stolon. It has an intertentacular organ. T. minini sp. nov. is the eleventh species described in the genus TriticellaDalyell, 1848, and the first record for this genus from the northwestern Pacific. The new species is the fifth ctenostome bryozoan known to occur in 5001-5500 m depth interval worldwide, and the deepest record reported for Triticella.

Unraveling micro- and nanoscale degradation processes during operation of high-temperature polymer-electrolyte-membrane fuel cells

NASA Astrophysics Data System (ADS)

Hengge, K.; Heinzl, C.; Perchthaler, M.; Varley, D.; Lochner, T.; Scheu, C.

2017-10-01

The work in hand presents an electron microscopy based in-depth study of micro- and nanoscale degradation processes that take place during the operation of high-temperature polymer-electrolyte-membrane fuel cells (HT-PEMFCs). Carbon supported Pt particles were used as cathodic catalyst material and the bimetallic, carbon supported Pt/Ru system was applied as anode. As membrane, cross-linked polybenzimidazole was used. Scanning electron microscopy analysis of cross-sections of as-prepared and long-term operated membrane-electrode-assemblies revealed insight into micrometer scale degradation processes: operation-caused catalyst redistribution and thinning of the membrane and electrodes. Transmission electron microscopy investigations were performed to unravel the nanometer scale phenomena: a band of Pt and Pt/Ru nanoparticles was detected in the membrane adjacent to the cathode catalyst layer. Quantification of the elemental composition of several individual nanoparticles and the overall band area revealed that they stem from both anode and cathode catalyst layers. The results presented do not demonstrate any catastrophic failure but rather intermediate states during fuel cell operation and indications to proceed with targeted HT-PEMFC optimization.

The rheo-Raman microscope: Simultaneous chemical, conformational, mechanical, and microstructural measures of soft materials

NASA Astrophysics Data System (ADS)

Kotula, Anthony P.; Meyer, Matthew W.; De Vito, Francesca; Plog, Jan; Hight Walker, Angela R.; Migler, Kalman B.

2016-10-01

The design and performance of an instrument capable of simultaneous Raman spectroscopy, rheology, and optical microscopy are described. The instrument couples a Raman spectrometer and optical microscope to a rotational rheometer through an optically transparent base, and the resulting simultaneous measurements are particularly advantageous in situations where flow properties vary due to either chemical or conformational changes in molecular structure, such as in crystallization, melting, gelation, or curing processes. Instrument performance is demonstrated on two material systems that show thermal transitions. First, we perform steady state rotational tests, Raman spectroscopy, and polarized reflection microscopy during a melting transition in a cosmetic emulsion. Second, we perform small amplitude oscillatory shear measurements along with Raman spectroscopy and polarized reflection microscopy during crystallization of a high density polyethylene. The instrument can be applied to study structure-property relationships in a variety of soft materials including thermoset resins, liquid crystalline materials, colloidal suspensions undergoing sol-gel processes, and biomacromolecules. Official contribution of the National Institute of Standards and Technology; not subject to copyright in the United States.

Fluorescence lifetime imaging microscopy using near-infrared contrast agents.

PubMed

Nothdurft, R; Sarder, P; Bloch, S; Culver, J; Achilefu, S

2012-08-01

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging. © 2012 The Author Journal of Microscopy © 2012 Royal Microscopical Society.

Functional biocompatible magnetite-cellulose nanocomposite fibrous networks: Characterization by fourier transformed infrared spectroscopy, X-ray powder diffraction and field emission scanning electron microscopy analysis.

PubMed

Habibi, Neda

2015-02-05

The preparation and characterization of functional biocompatible magnetite-cellulose nano-composite fibrous material is described. Magnetite-cellulose nano-composite was prepared by a combination of the solution-based formation of magnetic nano-particles and subsequent coating with amino celluloses. Characterization was accomplished using X-ray powder diffraction (XRD), fourier transformed infrared (FTIR) and field emission scanning electron microscopy (FESEM) analysis. The peaks of Fe3O4 in the XRD pattern of nanocomposite confirm existence of the nanoparticles in the amino cellulose matrix. Magnetite-cellulose particles exhibit an average diameter of roughly 33nm as demonstrated by field emission scanning electron microscopy. Magnetite nanoparticles were irregular spheres dispersed in the cellulose matrix. The vibration corresponding to the NCH3 functional group about 2850cm(-1) is assigned in the FTIR spectra. Functionalized magnetite-cellulose nano-composite polymers have a potential range of application as targeted drug delivery system in biomedical field. Copyright © 2014 Elsevier B.V. All rights reserved.