Ms Exposure Time Research Articles

Ultrabright and ultrastable near-infrared dye nanoparticles for in vitro and in vivo bioimaging

We report a new strategy of using carrier-free pure near-infrared (NIR) dye nanoparticles (NPs) to achieve highly luminescent NIR fluorescent probes for in vitro and in vivo imaging. Bis(4-(N-(2-naphthyl)phenylamino) phenyl)-fumaronitrile (NPAPF) NPs are shown to exhibit favorable biocompatibility, wide-range pH stability (pH 4–10) and much more superior photostability than conventional dyes. Importantly, the combined merits of high dye loading content and aggregation-induced emission enhancement properties, endow the NIR probes with high brightness and a high quantum yield up to 14.9%. The NPAPF NPs can be readily conjugated with folic acid for targeted in vitro cell imaging. Applications of the NPs probes in high efficiency in vivo and ex vivo imaging were successfully demonstrated. Intense fluorescent signals of NPAPF NPs can be distinctly, selectively and spatially resolved in tumor sites with ultrahigh sensitivity, even with 5 ms exposure time, due to the preferentially accumulation of NPs in tumor sites through passive enhanced permeability and retention effect. The totality of results clearly demonstrate the exciting potential of the functionalized NPAPF NPs as a NIR fluorescent probe for in vitro and in vivo imaging and diagnostics.

Toward the nanospring-based artificial olfactory system for trace-detection of flammable and explosive vapors

Chemical sensors were fabricated on the basis of novel nanomaterials – silica nanosprings. High chemical sensitivity was achieved by coating the silica nanosprings with ZnO using atomic layer deposition (ALD), followed by decorating with metal nanoparticles. The optimum operational conditions (T=400°C, ZnO grain size ∼15nm) were obtained and investigated. The nanospring-based sensors demonstrated remarkable vapor sensing properties: well-defined spikes in conductance upon exposure to explosives (TNT, TATP) and flammable vapors (toluene, acetone, ethanol) were obtained for 0.1ms exposure times at ppb level. Based on surface doping of ZnO with various metallic nanoparticles, a discrimination mechanism was developed and an integrated sensor-array for simultaneous real-time resistance scans was built. The integrated sensor response was tested using linear discriminant analysis (LDA). The distinguished electronic signatures of various chemical vapors were obtained at ppm level.

Widespread mRNA association with cytoskeletal motor proteins and identification and dynamics of myosin-associated mRNAs in S. cerevisiae.

Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe her e a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches.

ZnO coated nanospring-based chemiresistors

Chemiresistors were constructed using 3-D silica nanospring mats coated with a contiguous film of ZnO nanocrystals. Chemiresistors with an average ZnO nanocrystal radius <3 nm, or >20 nm, were found to exhibit a relative change in conductance of a factor of 50 upon exposure to a gas flow of 20% O2 and 80% N2 with ∼500 ppm of toluene and an operational temperature of 400 °C. Samples with an average ZnO nanocrystal radius of 15 nm were found to be the most responsive with a relative conductance change of a factor of 1000. The addition of metal nanoparticles (average radius equal to 2.4 nm) onto the surface of the ZnO nanocrystals (average radius equal to 15 nm) produced a relative change in conductance of a factor of 1500. For the optimum conditions (T = 400 °C, grain size ∼15 nm) well-defined spikes in conductance to explosive vapors (TNT, TATP) were obtained for 0.1 ms exposure time at ppb levels.

Experimental study of magnetic quantum-dot cellular automata function arrays

Magnetic quantum-dot cellular automata (MQCA) function arrays are fabricated by electron beam lithography, thermal evaporation and lift-off technologies at room temperature. The effects of exposure dose and exposure time on MQCA function array patterns with three various spacings are experimentally investigated. The results show that the ideal pattern can only be obtained with 100 pA electron beam current and 0.38 μs exposure time. Magnetic force microscopy measurement on the fabricated inverter structure shows that the array demonstrates correct logic operation, which validates the feasibility of fabrication process for MQCA function arrays. Moreover, defect is observed in the experiments, simulations on the defective array show that missing nanomagnet defect in the array leads to signal inversion error.

Revival of Prism-Based TIR Microscopy - Versatile Tracking of Fluorescent and Scattering Probes with NM-Precision

Current single-molecule microscopy experiments mostly rely on the fluorescence signal of fluorescent proteins, organic dyes or quantum dots attached to proteins of interest. However, these probes suffer from certain limitations, namely limited number of photons before photobleaching, photon blinking, and fluorescence saturation. Consequently, the temporal and spatial resolution by which single molecules can be tracked is limited. Promising candidates for replacing fluorescent probes are gold nanoparticles (GNPs). GNPs exhibit a large scattering cross section in the optical spectrum due to plasmon resonance, provide long-term stability and allow for versatile surface chemistry. Furthermore, because generation of photons relies on elastic scattering, their emission rate does not saturate.Here, we present a camera-based wide-field imaging technique for GNP-labeled proteins using a novel parabolic prism-type total-internal reflection (TIR) microscope. We demonstrate the advantages of GNPs over commonly used fluorescent probes and discuss the pros and cons of prism-type versus objective-type TIR microscopy. We demonstrate that prism-based TIR microscopy allows imaging of fluorescent and scattering probes with high signal-to-noise, excellent control over a wide range of incidence angles, and even illumination intensities within a field of view. When tracking surface-immobilized GNPs (40 nm diameter) we obtained two-dimensional localization precisions as good as 5 angstrom within 30 ms exposure time. We demonstrate localization experiments of kinesin-1 motors labeled with GNPs walking on surface-immobilized microtubules. When GNPs where bound to the tail or motor domains of kinesin-1 we found localization precisions of 3.6 nm and 1.9 nm within 30 ms exposure time, respectively. Furthermore GNP-loaded motors showed 8 nm stepping along microtubules at 3 μM MgATP within image acquisition times of 15 ms. Our method allows for precise localization of biomolecules within short acquisition times over long time scales.

Surface modifications of polymethylmetacrylate films using atmospheric pressure air dielectric barrier discharge plasma

In this paper, polymethylmetacrylate (PMMA) films are modified using an atmospheric pressure non-thermal plasma generated by a dielectric barrier discharge (DBD) in air. The DBD is generated in a plane-parallel reactor, which is driven by a μs pulse power supply with amplitude of up to 25 kV and repetition rate of 1 kHz, and the plasma generated shows homogeneous mode discharge characteristics verified by electrical measurements and light emission images with 0.5 ms exposure time. The treatment time ranging from 0 to 60 s and the discharge power density ranging from 11.62 to 30.83 W/m2 are used to study the effects of discharge parameters on the surface treatment, and the surface properties of PMMA films are studied using contact angle and surface energy measurement, scanning electron microscopy (SEM), atomic force microscopy (AFM), fourier transformed infrared spectroscopy (FTIR) and x-ray photoelectron spectroscopy (XPS). The study shows that, after the plasma treatment, the surface of PMMA film is etched, and oxygen-containing polar groups are introduced into the surface. These two processes can induce a remarkable decrease in water contact and a remarkable increase in surface energy, and the surface properties of PMMA films is improved accordingly. It is shown that the improvement of hydrophobicity depends on the discharge power density and treatment time, and there is a saturation treatment time at each discharge power density. Increasing discharge power density can induce more effective treatment of PMMA films, and less treatment time is needed to achieve the same level of surface treatment by increasing the discharge power density. Because more oxygen-containing polar groups are created and more obvious etching is occurred on the PMMA surface at higher discharge power density.

Subdiffraction-Limit Study of Kaede Diffusion and Spatial Distribution in Live Escherichia coli

Photoactivation localization microscopy (PALM) is used to study the spatial distribution and diffusion of single copies of the protein Kaede in the cytoplasm of live Escherichia coli under moderate growth conditions (67 min doubling time). The spatial distribution of Kaede is uniform within the cytoplasm. The cytoplasmic radius of 380 ± 30 nm varies little from cell to cell. Single-particle tracking using 4 ms exposure times reveals negatively curved plots of mean-square displacement versus time. A detailed comparison with Monte Carlo simulations in a spherocylindrical volume shows that the curvature can be quantitatively understood in terms of free diffusion within a confining volume. The mean diffusion coefficient across cells is <DKaede> = 7.3 ± 1.1 μm2·s−1, consistent with a homotetrameric form of Kaede. The distribution of squared displacements along the long axis for individual Kaede molecules is consistent with homogeneous diffusion. However, for longer cells, a spatial map of one-step estimates of the diffusion coefficient along x suggests that diffusion is ∼20–40% faster within nucleoids than in the ribosome-rich region lying between nucleoid lobes at the cell mid-plane. Fluorescence recovery after photobleaching yielded <DFRAP> = 8.3 ± 1.6 μm2·s−1, in agreement with the single-particle tracking results.

Measurement of Fluorescence in a Rhodamine-123 Doped Self-Assembled “Giant” Mesostructured Silica Sphere Using a Smartphone as Optical Hardware

The blue OLED emission from a mobile phone was characterised, revealing a sharp emission band centred at λ = 445 nm with a 3dB bandwidth Δλ ∼ 20 nm. It was used to excite Rhodamine 123 doped within a “giant” mesostructured silica sphere during fabrication through evaporative self-assembly of silica nanoparticles. Fluorescence was able to be detected using a standard optical microscope fitted with a green transmission pass filter and cooled CCD and with 1 ms exposure time demonstrating the potential of mobile platforms as the basis for portable diagnostics in the field.

Spectral (600–1050 nm) time exposures (99.6μs) of a lightning stepped leader

[1] A cloud-to-ground lightning stepped leader has been recorded with a slitless spectrograph at a recording rate of 10,000 images per second at a distance of 0.6 km. Five sequential images of the leader spectra were recorded with an exposure (integration) time of 99.6 μs each over a spectral range from 600 to 1050 nm. These are the first stepped leader spectra covering the range 600 to 1050 nm. The last three spectra, obtained immediately before the return stroke, were analyzed at an altitude of between 108 and 122 m above a struck vehicle. The spectral emissions in the near infrared are dominated by neutral nitrogen and oxygen emissions, and Hα, with only a few emission lines from singly ionized nitrogen. A singly ionized nitrogen line at 661.1 nm is present in the first analyzed image, but not in the two subsequent images at the same height, which suggests a cooling of the channel. The emissions are integrated over a 99.6 μs exposure time and therefore show no evidence of stepping. The ensuing negative return stroke was detected by the National Lightning Detection Network and had an estimated peak current of −15.2 kA. One subsequent stroke was outside the field of view of the spectrograph. The flash occurred on 11 September 2009 near New Underwood, South Dakota, and the exact location of the first stroke is known because it struck a car traveling on Interstate 90. The stepped leader two-dimensional speed increased in the last four steps from 1.53 × 105 to 2.42 × 105 m/s with an average of 2.03 × 105 m/s.

Design, characterization and evaluation of high performance 2.8 μm pitch zero space microlens

Utilization of the zero space microlens technology can significantly improve the image quality of CMOS sensors. In this study, we present systematical data of design, simulation, characterization and silicon level testing during the initial stage of development of the zero space microlens based CMOS imaging technology. The optimal structure of zero space microlens was obtained based on the simulation results. Sample CMOS image sensors with a 2.8 μm pitch zero space microlens above each pixel have been successfully fabricated based on 0.18 μm CMOS technology. Using AFM (atomic force microscopy) and sensor test platform, the structural and optical properties of both space microlens and zero space microlens have been characterized, and their performances have been evaluated respectively. Both AFM results and silicon tests have demonstrated that the 2.8 μm pitch zero space microlens can remarkably improve the pixel sensitivity and pixel array non-uniformity, and reduce the optical crosstalk. Compared to the space 2.8 μm square microlens, the zero space microlens shows 78.83% (68.42% and 75.93%) enhancement of photosensitivity and increment of pixel non-uniformity up to 20% (45.6% and 30.77%) for R (G and B), and reduction of the optical crosstalk up to 44.49%, under 45 lux light and 30 ms exposure time. In addition, the zero space microlens has also shown a great potential in further reducing pixel size down to less than 2.8 μm and meanwhile improving imaging performance of CMOS image sensors.

Ultrafast 3-D shape measurement with an off-the-shelf DLP projector

This paper presents a technique that reaches 3-D shape measurement speed beyond the digital-light-processing (DLP) projector's projection speed. In particular, a "solid-state" binary structured pattern is generated with each micro-mirror pixel always being at one status (ON or OFF). By this means, any time segment of projection can represent the whole signal, thus the exposure time can be shorter than the projection time. A sinusoidal fringe pattern is generated by properly defocusing a binary one, and the Fourier fringe analysis means is used for 3-D shape recovery. We have successfully reached 4,000 Hz rate (80 μs exposure time) 3-D shape measurement speed with an off-the-shelf DLP projector.

Observation of an Ablating Surface in Expansion Tunnel Flow

The observation of an ablating surface in expansion tunnel flow was reported. A one-dimensional (1-D) semi-infinite analysis was performed using an empirical estimation of the stagnation-point heat flux. A surface temperature change for epoxy of 178 K in 50 μs was calculated, which is sufficient for the epoxy coating to commence ablation during the steady test period. The 10% temperature penetration depth is 6 μm in 50 μs. The luminosity from substantial portions of the model and shock-layer flow was visualized using a Shimadzu HPV1 high-speed charge-coupled device (CCD) video camera recording at 500 kfps with a 1 μs exposure time. The uniform image response over the axisymmetric model implies the shock-layer gas irradiance is high immediately behind the shock and decreases rapidly as the model surface is approached. The measurements showed that use of an epoxy coating results in greatly increased CN and C2 radiation, much greater than when no coating is employed.

X-ray μCT imaging technique reveals corm microstructures of an arctic-boreal cotton-sedge, Eriophorum vaginatum

X-ray computed tomography (CT), a non-destructive imaging technique, has recently been effectively applied to botanical research. In this study an X-ray microCT technique was developed to allow for anatomical study of the overwintering corms of Eriophorum vaginatum, an ecologically important sedge species in arctic tussock-tundra and boreal peatlands. Using a GE Medical MS8X-130 X-ray microCT scanner, optimal imaging parameters included scanning isolated corms at 80 k Vp and 100 microA with a 3500 ms exposure time and an isotropic voxel size of 10 microm. A Gaussian blur image filter with a blur radius (sigma) of two pixels was applied to the optimal dataset to improve visual detection and contrast of tissues while removing 99.2% of image noise. Using the developed X-ray microCT technique several undocumented anatomical characteristics of the corm were identified including the vascular connection between a parent corm and branching cormel and the 3D shape of sclereid clusters. The 3D structure of sclereid clusters was determined whereby the perimeter of their lance shape is greatly reinforced by sclereids with thicker secondary cell walls as compared to those of the interior of the cluster. The structure of sclereid clusters and their association with leaf traces suggests they may be stabilizing the corm-leaf connection to protect vascular tissues from physical damage. The proposed X-ray microCT technique is an excellent tool for determination of the 3D structure of E. vaginatum corms and may be used to detect alterations in tissue structure and chemistry in response to environmental change in this and other Cyperaceous species.

Abstract LB-277: Abnormal lymphatic drainage pathway and function in mice with lymph node metastases

Abstract The lymphatic system provides a major route of cancer cell dissemination from the primary lesion site, to regional draining lymph nodes (LNs), through the lymphatic to blood vasculatures, and ultimately to distant sites of metastasis. However, to date there has been no in vivo imaging technique available to demonstrate functional and structural changes of overall lymphatic system in response to tumor growth and metastasis. In order to elucidate whether there are spatial and temporal changes in lymphatic function and architecture during tumor progression, we employed a dynamic near-infrared (NIR) fluorescence imaging technique. C6/Lacz rat glioma cells were implanted intradermally into the tail at 1 to 2 cm caudal to the rectum or in the right hindlimb in balb/c nude mice. Mice with a tumor in the tail were imaged at three weeks post implantation and mice with a tumor in the hindlimb at two and three weeks after tumor inoculation. Dynamic NIR imaging with 200 ms exposure time was conducted after intradermal injection of indocyanine green (ICG) to the base of the mouse tail for a hindlimb tumor model and to the tail 2cm away from the tail tip for a tail tumor model. Our imaging data in mice with a tail tumor show no uptake of fluorophore in the lymphatic capillaries in the skin above the tumor, which is in agreement with previous reports. In addition, our results show greater staining of lymphatic capillary network to the distal tumor periphery, dilated and tortuous collecting lymphatic vessels, and reduced or loss of propulsive lymphatic flow. Moreover, altered lymphatic drainage patterns and abnormal lymphatic flow from the collecting lymphatic vessel to the lymphatic capillaries at the distal tumor margin were detected. In a hindlimb tumor model, the lymphatic drainage pattern transiently changed with progressive disease. Mice with either the inguinal (primary) or axillary (secondary) LN metastasis showed dilated and leaky fluorescent lymphatic vessels, which circumvented the primary tumor and often the inguinal LN with a tumor and drained to the brachial LN. In one mouse with the inguinal and axillary LN metastases, ICG-labeled lymph drained across the midline of the animal body and thus to the left axillary LN. Magnified fluorescent images also show diffuse dye patterns. In addition, reduced lymphatic contractile function was seen in mice with LN metastasis. In summary, we showed in an animal model that gross cancer metastasis to the draining LNs is accompanied by abnormal lymphatic drainage pathway as well as reduced functional lymph propulsion in comparison to tumor-free mice. Therefore, this emerging NIR fluorescence imaging technology can be used to non-invasively and quantitatively detect functional lymphatic changes associated with cancer, which may enable accurate nodal staging of cancer patients and provide new approaches to diagnoses and therapies against cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-277.

Wide-field four-channel fluorescence imager for biological applications

A wide-field four-channel fluorescence imager has been developed. The instrument uses four expanded laser beams to image a large section (6 mm x 9 mm). An object can be sequentially illuminated with any combination of 408-, 532-, 658-, and 784-nm lasers for arbitrary (down to 1 ms) exposure times for each laser. Just two notch filters block scattered light from all four lasers. The design approach described here offers great flexibility in treatment of objects, very good sensitivity, and a wide field of view at low cost. There appears to be no commercial instrument capable of simultaneous fluorescence imaging of a wide field of view with four-laser excitation. Some possible applications are following events such as flow and mixing in microchannel systems, the transmission of biological signals across a culture, and following simulations of biological membrane diffusion. It can also be used in DNA sequencing by synthesis to follow the progress of the photolytic removal of dye and terminator. Without utilizing its time resolution, it can be used to obtain four independent images of a single tissue section stained with four targeting agents, with each coupled to a different dye matching one of the lasers.

High current diffuse dielectric barrier discharge in atmospheric pressure air for the deposition of thin silica-like films

The diffuse dielectric barrier discharge in atmospheric pressure air was applied for the thin film deposition on polymeric web in industrially relevant roll-to-roll configuration. The silica-like film deposition was performed using the admixture of hexamethyldisiloxane precursor to air flow. Fast discharge imaging at 2 μs exposure time confirms plasma uniformity in a single current pulse time scale. Morphology and composition analyses indicate that the process results in ultrasmooth films (roughness comparable to initial substrate roughness) and shows the possibility to synthesize carbon-free layers.

CW laser-induced fluorescence of toluene for time-resolved imaging of gaseous flows

A laser-induced fluorescence diagnostic is presented for high-speed measurements in gaseous flows. The technique employs a toluene tracer excited at 266 nm by a cavity-doubled 532 nm diode-pumped 5.5 W CW laser. The high power (600 mW) of UV light produced by cavity doubling, together with the high fluorescence yield of toluene, yields strong signal levels needed for high-speed recording. Fluctuation detection limits for tracer mole fraction were investigated by applying the diagnostic to an atmospheric temperature and pressure nitrogen jet. For single-point measurements with a photomultiplier tube, the detection limit for fluctuations in the toluene mole fraction was 0.028%, achieved with 430 mW of laser power and 8.5 kHz bandwidth for a 1×0.4×0.4 mm collection volume. Line (1-D) imaging with a kinetic-readout camera (512 pixels/row) achieved a detection limit of 0.23% with 440 mW of laser power, 9.7 kHz frame rate, and 0.3×0.2×0.4 mm collection volume per pixel, while planar (2-D) imaging with a 512×512 pixel intensified camera achieved a detection limit of 0.88% with 205 mW of laser power, 100 μs exposure time, and 0.4×0.4×0.4 mm volume per pixel. Line and planar imaging were applied to a turbulent jet with Re of about 10000.

Synchrotron-based radioscopy employing spatio-temporal micro-resolution for studying fast phenomena in liquid metal foams

Investigations of pore coalescence and individual cell wall collapse in an expanding liquid metal foam by means of X-ray radioscopy with spatio-temporal micro-resolution are reported. By using white synchrotron radiation for imaging, the rupture of a film and the subsequent merger of two neighbouring bubbles could be recorded with a time sampling rate of 40000 frames s(-1) (25 micros exposure time) and a spatial sampling rate of 20 microm. The rupture time of a cell wall was found to be in the range of 300 micros. This value is in agreement with theoretical considerations which assume an inertia-dominated rupture time of cell walls in liquid metal foams.

Adeno-associated Virus of a Single-polarity DNA Genome Is Capable of Transduction In Vivo

The adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. AAV vectors with wild-type inverted terminal repeats package either the plus- or the minus-strand DNA genomes with equal frequency. By creating a series of deletions within the, we have developed a genetic approach that can generate an AAV vector that packages its single-stranded DNA genome predominantly in a single polarity (99.4%). This novel reagent efficiently transduced muscle, brain and liver in whole animals. The transduction efficiencies were similar to those of the control mixed-polarity vectors. Our results showed that reannealing of plus- and minus-strand DNA was not required for AAV-mediated transduction in vivo, supporting the hypothesis that second-strand DNA synthesis is a primary pathway in converting the single-stranded AAV genome into double-stranded forms. The availability of the single-polarity AAV vector would aid further studies on the mechanism of AAV transduction as well as the application of AAV vector for gene replacement therapy.