Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging

A novel microfluidic approach for the quantification of reaction kinetics is presented. A three-dimensional finite difference numerical simulation was developed in order to extract quantitative kinetic information from fluorescence lifetime imaging experimental data. This approach was first utilized for the study of a fluorescence quenching reaction within a microchannel; the lifetime of a fluorophore was used to map the diffusion of a quencher across the microchannel. The approach was then applied to a more complex chemical reaction between a fluorescent amine and an acid chloride, via numerical simulation the bimolecular rate constant for this reaction was obtained.     

Metal-enhanced single-molecule fluorescence on silver particle monomer and dimer: coupling effect between metal particles

We prepared silver particle dimers with single Cy5 molecules localized between coupled metal particles. The silver particles with a 20 nm diameter were chemically bound with single-stranded oligonucleotides. The dimers were formed by hybridization with double-length single-stranded oligonucleotides that contained single Cy5 molecules. The image analysis revealed that the single-molecule fluorescence was enhanced 7-fold on the metal monomer and 13-fold on the metal dimer relative to the free Cy5-labeled oligonucleotide in the absence of metal. The lifetimes were shortened on the silver monomers and further shortened on the silver dimers, demonstrating the near-field interaction mechanism of fluorophore with the metal substrate. Finite-difference time-domain (FDTD) calculations were employed to study the distribution of electric field near the metal monomer and dimer. The coupling effect of metal particle on the fluorescence enhancement was discussed.     

Quantitative 3D mapping of fluidic temperatures within microchannel networks using fluorescence lifetime imaging

We describe a novel method for quantitatively mapping fluidic temperature with high spatial resolution within microchannels using fluorescence lifetime imaging in an optically sectioning microscope. Unlike intensity-based measurements, this approach is independent of experimental parameters, such as dye concentration and excitation/detection efficiency, thereby facilitating quantitative temperature mapping. Micrometer spatial resolution of 3D temperature distributions is readily achieved with an optical sectioning approach based on two-photon excitation. We demonstrate this technique for mapping of temperature variations across a microfluidic chip under different heating profiles and for mapping of the 3D temperature distribution across a single microchannel under applied flow conditions. This technique allows optimization of the chip design for miniaturized processes, such as on-chip PCR, for which precise temperature control is important.     

Phosphorescence lifetime imaging in turbid media: the inverse problem and experimental image reconstruction

Three-dimensional phosphorescence lifetime imaging is a novel method for the mapping of oxygen concentration in biological tissues. We present reconstruction techniques for recovering phosphorescent objects in highly scattering media based on the telegraph equation and two regularization methods, i.e., the Tikhonov-Phillips regularization and the maximum entropy method. Theoretical results are experimentally validated, and the reconstructed images of phosphorescent objects rendering oxygen maps in a layer are presented.     

Magnetic-nanoparticle-doped carbogenic nanocomposite: an effective magnetic resonance/fluorescence multimodal imaging probe

A novel and facile approach is developed to synthesize a magnetic nanoparticle (iron oxide)-doped carbogenic nanocomposite (IO-CNC) for magnetic resonance (MR)/fluorescence imaging applications. IO-CNC is synthesized by thermal decomposition of organic precursors in the presence of Fe(3) O(4) nanoparticles with an average size of 6 nm. IO-CNC shows wavelength-tunable fluorescence properties with high quantum yield. Magnetic studies confirm the superparamagnetic nature of IO-CNC at room temperature. IO-CNC shows MR contrast behavior by affecting the proton relaxation phenomena. The measured longitudinal (r(1) ) and transverse (r(2) ) relaxivity values are 4.52 and 34.75 mM(-1) s(-1) , respectively. No apparent cytotoxicity is observed and the nanocomposite shows a biocompatible nature. In vivo MR studies show both T(1) and T(2) * contrast behavior of the nanocomposite. Fluorescence imaging indicates selective uptake of IO-CNC by macrophages in spleen.     

Studying biological tissue with fluorescence lifetime imaging: microscopy,endoscopy, and complex decay profiles

We have applied fluorescence lifetime imaging (FLIM) to the autofluorescence of different kinds of biological tissue in vitro, including animal tissue sections and knee joints as well as human teeth, obtaining two-dimensional maps with functional contrast. We find that fluorescence decay profiles of biological tissue are well described by the stretched exponential function (StrEF), which can represent the complex nature of tissue. The StrEF yields a continuous distribution of fluorescence lifetimes, which can be extracted with an inverse Laplace transformation, and additional information is provided by the width of the distribution. Our experimental results from FLIM microscopy in combination with the StrEF analysis indicate that this technique is ready for clinical deployment, including portability that is through the use of a compact picosecond diode laser as the excitation source. The results obtained with our FLIM endoscope successfully demonstrated the viability of this modality, though they need further optimization. We expect a custom-designed endoscope with optimized illumination and detection efficiencies to provide significantly improved performance.     

UV fluorescence lifetime imaging microscopy: a label-free method for detection and quantification of protein interactions

Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 +/- 0.6) x 10(9) M(-1), represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays.     

Enhanced fluorescence of Cy5-labeled DNA tethered to silver island films: fluorescence images and time-resolved studies using single-molecule spectroscopy

Methods that increase the total emission per fluorophore would provide increased sensitivity and a wider dynamic range for chemical analysis, medical diagnostics, and in vivo molecular imaging. The use of fluorophore-metal interactions has the potential to dramatically increase the detectability of single fluorophores for bioanalytical monitoring. The fabrication and single-molecule analysis of fluorophore-labeled DNA molecules tethered to silver island films are described in this article. The single-molecule spectroscopic method reveals some insightful information on the behaviors of single molecules, rather than an ensemble of molecules. Analysis of fluorescence images, intensity profiles, total emitted photons, and lifetime distributions reveals some of sample heterogeneities. Investigations of time-dependent emission characteristics of single molecules indicate that the total number of emitted photons on the silvered surface is more than 10 times greater than on free labeled DNA molecules on a glass substrate. In addition, time-correlated single-photon counting results reveal the reduced lifetimes of single molecules tethered to silver island films.     

An assay of ganglioside using fluorescence image analysis on a thin-layer chromatography plate

A new and simple fluorometric method for determine quantities of gangliosides ranging from pico- to nanomoles is reported. Spraying hydrochloric acid followed by a heating treatment, sugars (glucose, galactose, fucose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid (sialic acid)), gangliosides (G(M1), G(D1a), G(T1b)), and asialoganglioside (asialoG(M1)) on thin-layer chromatography plates produced fluorescence under 365-nm UV light. This fluorescence production of each sample was greatly dependent on the heating temperature. As sialic acid fluoresced readily at lower temperature (approximately 90 degrees C), we were able to distinguish sialic acid easily from other sugars tested. To determine gangliosides based on this sialic acid fluorescence, calibration curves for gangliosides were obtained by high-performance thin-layer chromatography and the image-analyzing system equipped with a CCD camera. The observed fluorescence images were analyzed using image-analyzing software packages and the determined calibration curves for ganglioside-bound sialic acids were reproducible and showed a high linearity in a wide range from 47 pmol to 4.5 nmol. Since the fluorescence from sialic acid is easily measurable on TLC plates and is sensitive over a wide range of sample concentration, the present method is applicable for quantitative determination of gangliosides.     

A new method for quantitative cellular imaging on 3-D scaffolds using fluorescence microscopy

This paper presents a new image processing technique for estimating cell numbers and contours in three-dimensional (3-D) microfabricated scaffolds. The method is based on a statistical approach, and utilizes the extreme value theory, which assumes that cells in the image field are rare, high-intensity events. Confocal microscopy images of fibroblasts on 3-D structures were processed using the method, and the resulting data on cell numbers was compared with countings obtained using a Burker chamber. The results were identical to within a few percent.     

Fabrication of Bi2Sr2CaCu2Ox films on metal substrates by the chemical process using metal alkoxides

The formation behaviour of Bi₂Sr₂CaCu₂O_x from compounds prepared by hydrolysis of metal alkoxides was studied and Bi₂Sr₂CaCu₂O_x films on metal substrates were fabricated using a metal alkoxide solution. Bi₂Sr₂CaCu₂O_x was formed through intermediate phases such as Bi₂Sr₂Cu₁O_x, Bi₂CuO₄, SrCO₃, CaCO₃ and CuO. Bi₂CuO₄ was initially formed with SrCO₃, CaCO₃ and CuO, and then reacted with SrCO₃ to form Bi₂Sr₂Cu₁O_x. Bi₂Sr₂Cu₁O_x reacted with CaCO₃ and CuO to give Bi₂Sr₂CaCu₂O_x. Bi₂Sr₂CaCu₂O_x films were successfully fabricated on nickel substrates using the metal alkoxide solution at the nominal composition of BiSrCaCu=2223. Bi₂Sr₂CaCu₂O_x was precipitated on Ni substrates at firing temperature of 770 °C or above, and a sharp T _c was obtained at the firing temperature of 800 °C.     

Verification of antiparallel G-quadruplex structure in human telomeres by using two-photon excitation fluorescence lifetime imaging microscopy of the 3,6-Bis(1-methyl-4-vinylpyridinium)carbazole diiodide molecule

Different G-quadruplex structures for the human telomeric sequence d(T2AG3)4 in vitro have been documented in the presence of sodium and potassium. Verification of the G-quadruplex structures in human telomeres in vivo is the main issue in establishing the biological function of the G-quadruplex structures in telomeres as well as the development of anticancer agents. Here we have applied two-photon excitation fluorescence lifetime imaging microscopy to measure the fluorescence lifetime of the BMVC molecule upon interaction with various DNA structures. The distinction in lifetime measured with submicrometer spatial resolution in two-photon excitation fluorescence lifetime imaging microscopy provides a powerful approach not only to verify the existence of the antiparallel G-quadruplex structure in human telomeres but also to map its localizations in metaphase chromosomes.     

Real-time heterodyne imaging interferometry: focal-plane amplitude and phase demodulation using a three-phase correlation image sensor

A method of real-time heterodyne imaging interferometry using a three-phase correlation image sensor (3PCIS) is proposed. It simultaneously demodulates the amplitude and phase images of an incident interference pattern at an ordinary frame rate with good accuracy, thus overcoming the trade-off among measurement time, spatial resolution, and demodulation accuracy suffered in conventional interferometry. An experimental system is constructed with a 64x64 3PCIS camera operated at 30 frames/s and a two-frequency He-Ne laser with a beat frequency of 25 kHz. The results obtained for a scanning mirror and heated silicone oil confirm the proposed method.     

A survey on performance status of mammography machines: image quality and dosimetry studies using a standard mammography imaging phantom

It is essential to perform quality control (QC) tests on mammography equipment in order to produce an appropriate image quality at a lower radiation dose to patients. Imaging and dosimetric measurements on 15 mammography machines located at the busiest radiology centres of Mumbai, India were carried out using a standard CIRS breast imaging phantom in order to see the level of image quality and breast doses. The QC tests include evaluations of image quality and the mean glandular doses (MGD), which is derived from the breast entrance exposure, half-value layer (HVL), compressed breast thickness (CBT) and breast tissue compositions. At the majority of the centres, film-processing and darkroom conditions were not found to be maintained, which is required to meet the technical development specifications for the mammography film in use as recommended by the American College of Radiology (ACR). In most of the surveyed centres, the viewbox luminance and room illuminance conditions were not found to be in line with the mammography requirements recommended by the ACR. The measured HVL values of the machines were in the range of 0.27-0.39 mm aluminium (Al) with a mean value of 0.33±0.04 mm Al at 28 kV(p) following the recommendation provided by ACR. The measured MGDs were in the range of 0.14-3.80 mGy with a mean value of 1.34 mGy. The measured MGDs vary between centre to centre by a factor of 27.14. Referring to patient doses and image quality, it was observed that only one mammography centre has exceeded the recommended MGD, i.e. 3.0 mGy per view with the value of 3.80 mGy and at eight mammography centres the measured central background density (CBD) values for mammography phantom image are found to be less than the recommended CBD limit value of 1.2-2.0 optical density.     

纳米Y2O2S∶Eu的燃烧法合成和Eu3+离子荧光探针物相分析

将硝酸钇铕和较便宜的水溶性含硫燃烧剂溶于乙醇-水溶液,通过新的乙醇辅助燃烧法合成了一次粒径约20nm的硫氧化钇(Y2O2S∶Eu)发光材料.研究结果表明:采用硫代乙酰胺作为燃烧剂时,燃烧反应产物为Y2O2S∶Eu及微量的Y2O3∶Eu.而采用硫脲作为燃烧剂,燃烧反应产物为Y2O3∶Eu,Y2O2S∶Eu和/或Y2O2SO4 ∶Eu的混合物.利用Eu3 离子的5D0→7Fj跃迁导致的红色发光作为荧光探针,根据其在Y2O3(λem=610nm),Y2O2S(λem1=625nm,λem2=615nm),Y2O2SO4(λem1=613,λem2=615nm)基质中的光致发光光谱和X射线发光光谱位置和强度不同,来确定生成物中Y2O3∶Eu,Y2O2S∶Eu,Y2O2SO4 ∶Eu物相的组成.     

Direct imaging of molecular orbitals of metal phthalocyanines on metal surfaces with an O2-functionalized tip of a scanning tunneling microscope

High-resolution scanning tunneling microscope images of iron phthalocyanine and zinc phthalocyanine molecules on Au(111) have been obtained using a functionalized tip of a scanning tunneling microscope (STM), and show rich intramolecular features that are not observed using clean tips. Ab initio density functional theory calculations and extended Hückel theory calculations revealed that the imaging of detailed electronic states is due specifically to the decoration of the STM tip with O₂. The detailed structures are differentiated only when interacting with the highly directional orbitals of the oxygen molecules adsorbed on a truncated, [111]-oriented tungsten tip. Our results indicate a method for increasing the resolution in generic scans and thus, have potential applications in fundamental research based on high-resolution electronic states of molecules on metals, concerning, for example, chemical reactions, and catalysis mechanisms.     

Analysis of Al/Al2O3 metal matrix composites under biaxial cyclic loading using a digital image based finite element method

Abstract

This paper examines the use of a two­dimensional digital image based finite element method to predict the global behaviour of multiphase material systems. Micrographic images are digitised and meshed for implementation into the general purpose finite element code ADINA. The global cyclic response of the composite can be effectively modelled by using an appropriate constitutive relationship to describe the cyclic elastic–plastic behaviour of the matrix phase. The main advantage of the digital image based method is that the actual microstructural details including particle size, shape, and distribution are inherently captured in the analysis. The predicted global stress–strain responses of aluminium alloy 6061-T0/Al₂O₃particulate metal matrix composites under uniaxial and biaxial loading conditions (monotonic and cyclic) are found to correlate accurately with experimental results. When compared with predictions based on existing unit cell models, a noticeable improvement is observed. The effect of the representative length­scale (field of view) used in the analysis was found to be quite important in determining an accurate global response. A statistical analysis using uniformly derived lineal fraction measurements was also performed to demonstrate the correlation between the particle morphology in a particular field of view and the measured global response. Preliminary results indicate that this analysis technique may provide a possible method for determining the appropriate length­scale for which global analysis applies.     

Applicability of X-ray fluorescence spectroscopy as method to determine thickness and composition of stacks of metal thin films: A comparison with imaging and profilometry

In this work the applicability of X-ray fluorescence spectroscopy (XRF) for fast, accurate and non-destructive determination of the thickness of a variety of single-layer and multi-layer metal thin films deposited on glass and silicon is investigated. Data obtained with XRF is compared with information from profilometry and images from scanning electron microscopy (SEM). Whereas thickness determinations based on profilometry and cross-sectional SEM-imaging have restrictions with respect to thickness of metal stacks or hardness of the metals, XRF has no such limitations. Moreover, XRF can discriminate between sublayers in a multi-layer film, and can also be utilized for compositional analysis and density estimations. Good agreement between thickness data obtained with XRF, profilometry and SEM-images is found, under the justifiable assumption that the density of sputter-deposited and evaporated thin films is ca. 5% below that of bulk metals. Similar XRF-results are found for non-patterned areas (64 mm² metal) as well as lithographically patterned areas containing a series of small metal lines (total metal surface ca. 8 mm²). As a consequence, it is concluded that XRF is a versatile technique for analysis, verification, control or evaluation of the thickness, density or (elemental) composition of thin metal film line-patterns, during their fabrication as well as prior or post to applications.     

Multimodal atomic force microscopy: Biological imaging using atomic force microscopy combined with light fluorescence and confocal microscopies and electrophysiologic recording

Because the atomic force microscope (AFM) allows molecular resolution imaging of hydrated specimens, it provides a unique window to the microscopic biological world. A high signal-to-noise ratio in AFM images sets them apart from the images obtained from other techniques: One does not need extensive image analyses often required by other techniques to obtain high-resolution information. AFM can provide molecular details on crystalline as well as amorphous materials. However, it is often limited in providing identity of the imaged structures, especially in a complex system such as a cellular membrane. AFM's application for biological imaging will rely on an unambiguous identification of imaged structures. For mixed macromolecules, it may be essential to make critical comparisons of the same structural features imaged with AFM and other techniques such as light fluorescence and confocal microscopies, electron microscopy and X-ray diffraction, and biochemical, immunologic, and pharmacologic techniques and electrophysiologic recordings. Significantly, the simple design of AFM allows it to be integrated with other techniques for simultaneous multimodal imaging. Recent combined multimodal imaging include light fluorescence, confocal, and near-field optical imaging as well as electrophysiologic recordings. Preliminary studies from such multimodal imaging include 1) an independent identification of macromolecules in a complex specimen using appropriately labeled markers such as fluorescent-dye labeled antibodies or dark-field microscopy; 2) imaging real-time reorganization of surface features using laser confocal and AFM; 3) a direct correlation of structural features and ion transfer via pores in a membrane; and 4) macromolecular complexes such as receptor-ligand and antigen-antibody. These features of a multimodal imaging system will provide new and significant avenues for a direct real-time structure-function correlation studies of biological macromolecules. © 1997 John Wiley & Sons, Inc. Int J Imaging Syst Technol, 8, 293–300, 1997