High-sensitivity Camera Research Articles

Dual-color prism-type TIRFM system for direct detection of single-biomolecules on nanoarray biochips

Abstract This study examined the applicability of a prism-type simultaneous dual-color total internal reflection fluorescence microscopy (TIRFM) system for the simultaneous detection of nano biomolecules on nanoarray biochips at the single-molecule level. The dual-color TIRFM system with two individual laser beams and a high-sensitivity camera was used for the simultaneous dual-color detection of two different nano beads (i.e. 20 nm yellow–green and crimson fluorescent FluoSpheres beads), and single-protein molecules labeled with different fluorescent dyes (i.e. actin from rabbit muscle conjugated with Alexa Fluor ® 488 and Alexa Fluor ® 633 goat anti-rabbit IgG) without a time-delay and the need to move the sample. When this system was applied to two different single-protein molecules labeled with different fluorescent dyes on the GPTS/CHI/GA-modified glass nanoarray chip, the full images of the biomolecules at the single-molecule level were obtained simultaneously in two different colors using a Dual-View™. The dual-color TIRFM system is quite suitable for the biological imaging at the single-molecule level on nanoarray biochips. This study provides a benchmark for directly monitoring the interactions and detecting the colocalization of two different nano biomolecules, and can be applied to the development of a nanoarray biochip at the single-molecule level.

Single-Protein Molecular Interactions on Polymer-Modified Glass Substrates for Nanoarray Chip Application Using Dual-Color TIRFM

The immobilization of proteins and their molecular interactions on various polymer-modified glass substrates [i.e. 3-aminopropyltriethoxysilane (APTS), 3-glycidoxypropyltrimethoxysilane (GPTS), poly (ethylene glycol) diacrylate (PEG-DA), chitosan (CHI), glutaraldehyde (GA), 3-(trichlorosilyl)propyl methacrylate (TPM), 3'-mercaptopropyltrimethoxysilane (MPTMS), glycidyl methacrylate (GMA) and poly-l-lysine (PL).] for potential applications in a nanoarray protein chip at the single-molecule level was evaluated using prismtype dual-color total internal reflection fluorescence microscopy (dual-color TIRFM). A dual-color TIRF microscope, which contained two individual laser beams and a single high-sensitivity camera, was used for the rapid and simultaneous dual-color detection of the interactions and colocalization of different proteins labeled with different fluorescent dyes such as Alexa Fluor ® 488, Qdot ® 525 and Alexa Fluor ® 633. Most of the polymer-modified glass substrates showed good stability and a relative high signal-to-noise (S/N) ratio over a 40-day period after making the substrates. The GPTS/CHI/GA-modified glass substrate showed a 13.5-56.3% higher relative S/N ratio than the other substrates. 1% Top-Block in 10 mM phosphate buffered saline (pH 7.4) showed a 99.2% increase in the blocking effect of non-specific adsorption. These results show that dual-color TIRFM is a powerful methodology for detecting proteins at the single-molecule level with potential applications in nanoarray chips or nano-biosensors.

Endoscopic identification and biopsy sampling of an intraventricular malignant glioma using a 5–aminolevulinic acid–induced protoporphyrin IX fluorescence imaging system

Several neurosurgical studies have provided descriptions of the utility of fluorescence-guided tumor resection using a microscope. However, fluorescence-guided endoscopic detection of a deep-seated brain tumor has not yet been reported. The authors report their experience with an endoscopic biopsy procedure for a malignant glioma within the third ventricle using a 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX fluorescence imaging system. A 5-ALA-induced fluorescence image of an intraventricular tumor is barely visible with the typical fluorescence endoscopic system used in other clinical fields because the intensity of excitation light at wavelengths of 390 to 405 nm through a cut-off filter is too weak to delineate a brain tumor. The technique described in this study made use of a laser illumination system with a high-powered output that delivered a violet-blue light at wavelengths of 405 nm. In addition, a common ultraviolet cutoff filter was fitted between the endoscope and the high-sensitivity camera to block the backscattered excitation light. A 5-ALA-induced fluorescence endoscopy performed using this system allowed the intraventricular tumor to be clearly visualized as a red fluorescent lesion. Several biopsy specimens obtained from the fluorescent lesion provided a definitive histological diagnosis. The results indicate that this endoscopic system is useful in detecting an intraventricular fluorescent tumor.

In vivo Real-time Tracking of Single Quantum Dots Conjugated with Monoclonal Anti-HER2 Antibody in Tumors of Mice

Studies with tracking of single nanoparticles are providing new insights into the interactions and processes involved in the transport of drug carriers in living mice. Here, we report the tracking of a single particle quantum dot (Qdot) conjugated with tumor-targeting antibody in tumors of living mice using a dorsal skinfold chamber and a high-speed confocal microscope with a high-sensitivity camera. Qdot labeled with the monoclonal anti-HER2 antibody was injected into mice with HER2-overexpressing breast cancer to analyze the molecular processes of its mechanistic delivery to the tumor. Movement of single complexes of the Qdot-antibody could be clearly observed at 30 frames/s inside the tumor through a dorsal skinfold chamber. We successfully identified six processes of delivery: initially in the circulation within a blood vessel, during extravasation, in the extracelullar region, binding to HER2 on the cell membrane, moving from the cell membrane to the perinuclear region, and in the perinuclear region. The six processes were quantitatively analyzed to understand the rate-limiting constraints on Qdot-antibody delivery. The movement of the complexes at each stage was "stop-and-go." The image analysis of the delivery processes of single particles in vivo provides valuable information on antibody-conjugated therapeutic nanoparticles, which will be useful in increasing therapeutic efficacy.

Head–disk spacing measurement based on Michelson interferometry using white light illumination

Head–disk spacing of hard disk drives was measured based on Michelson interferometry using white light illumination. This method utilizes the phase comparison of two interference fringe patterns formed respectively on the inner surface of a glass disk and the air bearing surface of a slider through the glass disk. To suppress interference noise and to further enhance measurement accuracy, low coherent white light was adopted as an illuminating source to replace high coherent laser. A high sensitive CCD digital camera was used to compensate for the attenuation of the brightness and contrast of the interference fringe pattern in the white light-based Michelson interferometer. The captured images and the measured results indicate that the application of white light illumination effectively improves the interference fringe pattern, and the measurement error of extracting the ridgeline can be consequently reduced five times in comparison with a laser. Spacing resolution (=λ/2w) was enhanced to 0.68 nm/pixel in our experiment, closely approaching the extreme spacing resolution (at the largest fringe interval wmax) of 0.64 nm/pixel. Indirect and direct methods for determining fringe interval were proposed, and the latter provides higher accuracy. Head–disk spacing was measured at different spacing resolutions of 2.61 and 0.68 nm/pixel to clarify the effect of fringe interval on measurement accuracy. The results show that tested spacing at larger fringe interval achieves not only better agreement with the calculated example, but also better deviation tolerance characteristics. Based on the advantages, measurement of 10 nm spacing was realized with high accuracy and good repeatability.

Fluorescence endoscopic video system

This paper describes a fluorescence endoscopic video system intended for the diagnosis of diseases of the internal organs. The system operates on the basis of two-channel recording of the video fluxes from a fluorescence channel and a reflected-light channel by means of a high-sensitivity monochrome television camera and a color camera, respectively. Examples are given of the application of the device in gastroenterology.

Highlights from the Literature

Highlights from the Literature

GMA溶接における溶滴の温度計測

Temperatures of metal drop in GMA short-circuiting welding process were in-situ measured using newly developed instrument designed on the basis of two-color pyrometry, which consisted of optical lenses, interference filters for two colors and two sets of high sensitive CCD cameras with fast shutter. In order to avoid radiation from arc plasma, temperature measurement was carried out immediately after molten drop at electrode wire tip was contacted with weld pool and arc was extinguished. Welding current in arcing period was adjusted from 50 A to 250 A using experimental power source in Ar + 20%CO2 mixture gas shielded GMA welding with mild steel wire of 1.2 mm in diameter. It is shown through in-situ measurement that average temperature of metal drop ranges from 2200 K to 2700 K, depending on level and period of arc current governing electrode wire melting.

Laser-induced fluorescence with an OPO system. Part I. Optimisation of the analytical system by use of experimental design methodology. Application to the direct quantification of traces of benzo[ a]pyrene.

This study deals with the optimisation and application of a method for direct analysis of trace pollutants in water by laser-induced fluorescence. The arrangement used consisted of an Nd:YAG Laser coupled with an optical parametric oscillator (LYOPO) and connected to a spectrophotometer and a high-sensitivity camera. Optimisation was achieved by developing an experimental design methodology to maximise the signal-to-noise ratio and reduce the limit of detection. The technique was then applied to the detection of benzo[ a]pyrene in water. The experimental results were evidence of its high sensitivity and time-resolution potential. The detection limit for benzo[ a]pyrene was 0.7 ng L(-1) in drinking water and 4 ng L(-1) in raw water containing 1 mg L(-1) humic acids.

X-Ray Film Photoroentgenography in Modern Medical Practice

The goal of this work was to discuss possible ways to reduce the dose load on patients during mass-scale X-ray examinations using film photoroentgenographs. There is a trend in medical roentgenography all over the world toward abandonment of photoroentgenographic methods in favor of conventional X-raying or modern digital methods of X-ray examination. However, because digital apparatuses are rather expensive and implement a radically new approach to photoroentgenographic examination, their introduction into practice will require several years. During this time, conventional film photoroentgenographs will still be used. Approximately 5600 film photoroentgenographs are currently used in domestic health service. New domestic 12F9 photoroentgenographs with high-sensitivity photoroentgenographic cameras KF-400 constitute about 50% of them; already out-dated domestic 12F7 photoroentgenographs with low-sensitivity photoroentgenographic KF-70 cameras constitute 40%; foreign photoroentgenographs with out-dated RK-70 and RK-110 cameras made in the German Democratic Republic, 10%. The main problem of mass-scale photoroentgenography is to reduce the dose load without decreasing the information value of photoroentgenographic images. To solve this problem, domestic 12F9 photoroentgenographs were equipped with modern domestic KF-400 cameras with high-quality input screens and improved optics compatible with both 70 and 100-mm films [1]. The use of a new type of output luminescent screen is the main advantage of the KF-400 photoroentgenographic camera. Until recently, the ERS-Ts-1A luminescent screen based on ZnCdS:Ag has been used in photoroentgenographic cameras. Presently, a new luminescent screen ERS-G-2V based on the rare-earth luminophore Gd2O2S:Tb is used in the KF-400 camera. The main parameters of the luminescent screens are given in Table 1. Table 1 shows that the gadolinium-based ERS-G2V screen provides better image quality than the other model, especially if the effective energy of X-ray radiation is higher than that used in conventional apparatuses. Such screens provide higher resolution and contrast sensitivity, which increases information content of images. Also, it was shown that in screens based on gadolinium luminophores the coefficient of correspondence between the spectral sensitivity of photoroentgenographic film and spectral characteristics of the luminescent screen radiation is approximately 25% higher than in other models. Thus, replacement of the ERS-Ts-1A screen with the ERS-G-2V screen increases the sensitivity of photoroentgenographic cameras more than twofold and the resolution from 2.0 to 2.5 lines/mm. The contrast sensitivity is also improved (2% instead of 3%). The KF-400 camera contains an objective lens with improved characteristics as compared with the previous model KF-70. The lens used in the KF-400 camera provides better light transmission. Its main advantage is that it provides significantly lower vignetting (decrease in brightness from the center of an image to its edges) than the KF-70 model. In the KF-70 camera, vignetting at a distance of 0.7 working diameter is 70%; at a distance of 0.9 working diameter, 90%; whereas in the KF-400 camera vignetting at a distance of 0.9 working diameter does not exceed 50%. Losses in resolution at the edges of an image are also decreased (not more than 20%). The effective frame size of the KF-400 camera is larger than in the previous model. As a consequence, it provides higher sensitivity and significantly higher information content of images.

LS1B3 一分子イメージング用高感度カメラ(1分子イメージングと高感度カメラの応用,ランチョンセミナー,日本生物物理学会第40回年会)

LS1B3 一分子イメージング用高感度カメラ(1分子イメージングと高感度カメラの応用,ランチョンセミナー,日本生物物理学会第40回年会)

A 1/2-in, 1.3 M-pixel progressive-scan CCD image sensor employing 0.25-μm gap single-layer poly-Si electrodes

A 1/2-in, 1.3 M-pixel progressive-scan interline-transfer charge coupled-device (IT-CCD) image sensor has been developed for low-power and high-sensitivity digital cameras. The image sensor uses 0.25-/spl mu/m gap single-layer poly-Si for CCD transfer electrodes in order to reduce the power consumption and number of fabrication process steps. The image sensor achieved a low driving voltage (2.1 V) on a horizontal CCD (H-CCD) at a frequency of 24.5 MHz. An original pixel layout and a self-aligned photodiode structure make it possible to achieve a progressive scan pixel with well-controlled photodiode readout characteristics. An output three-stage source follower amplifier with new multioxide transistors, whose gate insulator thickness is thinner than that of a CCD register, is able to attain 17% higher gain than that of the conventional amplifier. The sensor provides low-power (100 mW) and high output sensitivity. The total number of steps for fabricating the sensor was reduced to 70% of that for conventional three-layer poly-Si electrodes.

Response of intra-acinar pulmonary microvessels to hypoxia, hypercapnic acidosis, and isocapnic acidosis.

To elucidate the differential reactivity of pulmonary microvessels in the acini to hypoxia, excessive CO2, and increased H+, we investigated changes in the diameter of precapillary arterioles, postcapillary venules, and capillaries in isolated rat lungs on exposure to normocapnic hypoxia (2% O2), normoxic hypercapnia (15% CO2), and isocapnic acidosis (0.01 mol/L HCl). Microvascular diameters were precisely examined using a real-time confocal laser scanning luminescence microscope coupled to a high-sensitivity camera with an image intensifier. Measurements were made under conditions with and without indomethacin or N(omega)-nitro-L-arginine methyl ester to assess the importance of vasoactive substances produced by cyclooxygenase (COX) or NO synthase (NOS) as it relates to the reactivity of pulmonary microvessels to physiological stimuli. We found that acute hypoxia contracted precapillary arterioles that had diameters of 20 to 30 microm but did not constrict postcapillary venules of similar size. COX- and NOS-related vasoactive substances did not modulate hypoxia-elicited arteriolar constriction. Hypercapnia induced a distinct venular dilatation closely associated with vasodilators produced by COX but not by NOS. Arterioles were appreciably constricted in isocapnic acidosis when NOS, but not COX, was suppressed, whereas venules showed no constrictive response even when both enzymes were inhibited. Capillaries were neither constricted nor dilated under any experimental conditions. These findings suggest that reactivity to hypoxia, CO2, and H+ is not qualitatively similar among intra-acinar microvessels, in which COX- and NOS-associated vasoactive substances function differently.

A miniature CCD video camera for high-sensitivity light measurements in freely behaving animals

We developed a miniaturized, high-sensitivity camera that can be placed in areas of difficult access in freely behaving animals for neural tissue imaging. The device consists of a charged coupled device (CCD) chip, a coherent image conduit and miniature light emitting diodes (LEDs). An amplifier circuit is constructed on the camera chip and nine wires are attached for external connections. Placement of LEDs around the image conduit perimeter provides dark-field illumination, which increases detection of cellular-related light scattering changes and doubles the depth-of-view over conventional reflectance imaging procedures. The device has been successfully used to record from several deep brain structures, including the ventral medullary surface of sleeping and waking cats. The procedure allows assessment of light scattering changes that result from neural activity or detection of vital dyes to metabolic or voltage-induced activation.

Design of a high-resolution, high-sensitivity PET camera for human brains and small animals

The authors present design parameters for a 3-D PET camera with high sensitivity (35 cm detector ring diameter, 15 cm axial field of view) and isotropic high resolution provided by detector modules capable of depth of interaction (DOI) measurement. Detector modules are made of LSO crystals (3 mm square by 30 mm deep)-the small module size and short decay time of LSO reduce the detector dead time by a factor of 14 compared to conventional EGO detector modules and narrow the coincidence window width to 4 ns. This yields an expected peak noise equivalent count rate of 800 kcps and noise equivalent sensitivity of 1370 kcps//spl mu/Ci/cc with a 20 cm diameter phantom-three to five times higher than conventional scanners. With 5 mm fwhm DOI resolution, the expected reconstructed spatial resolution is <3.0 mm fwhm throughout the entire field of view. Depth of interaction measurement information is incorporated into the reconstruction algorithm by rebinning onto a regularly spaced grid. Attenuation correction is performed with an orbiting single transmission source.

Time-Resolved High-Resolution Electron Microscopy of Clusters, Surfaces, and Interfaces

Nanoscale materials show a variety of interesting physical and chemical properties. Since the properties are mostly determined by mesoscopic- and atomicscale areas of the materials, characterization of nanoscale materials at the atomic level is crucial for understanding the origin of their properties as well as for tailoring them to industrial needs. High-resolution electron microscopy (HREM) is an effective method to observe atomic structures in nanophase materials. The “structure-imaging technique,” originated by Ueda et al., and Cowley and Iijima, contributed much to the development of the method. Innovations are anticipated in (1) three-dimensional analysis of nanophase materials in real space, (2) observation of dynamic phenomena, and (3) extraction of compositional and physical information from nanometer-sized areas. Using the structure-imaging technique, one can observe directly nanometer-scale particles and defect structures such as voids and line and plane defects in crystals, including surfaces and interfaces. There were previously some limitations on the study of the dynamic process on the atomic level since images were captured on films. The recent development of high-sensitivity silicon-integrated-target-television (SIT-TV) cameras has initiated a new stage of HREM.Our time-resolved-high-resolution-electron-microscopy (TRHREM) system is composed of a 200-kV HREM (JEOL: JEM-2010) equipped with a high-sensitivity SIT-TV camera, various types of dedicated sample holders, an ultrahigh-vacuum (UHV) sample-preparation chamber, and a digital videotape recorder, as partly illustrated in Figure 5. The HREM images at 0.8–1.0-million magnification are recorded on the videotape recorder. In the U.S.-Japan TV system, one “frame image” of 1/30-s time resolution is composed of two interlaced “field images” of 1/60-s time resolution. The digital videotape recorder enables us to output the two “field images” separately from two frame memories.

Time-resolved high-resolution electron microscopy of structural transformation in nanocrystalline ZnO films

Mechanical properties of polycrystalline materials become anomalous when the grain size and grain boundary length decrease to nanometer scale. For example, ductility and toughness increase significantly in nanometer-grained ceramics (nanocrystalline ceramics). Ductility increases due to appearance of fine-grained-superplastic deformation. Grain boundary migration and interface migration are fundamental processes of the superplastic deformation. Structural transformation of fine grains is a factor which limits the toughness in polycrystalline ceramics because the transformation relaxes internal strain. The behavior of grain boundaries and interfaces, such as diffusion bonding and Czochralski-type crystal growth at ambient temperature, can be analyzed by a time-resolved high-resolution electron microscopy (TRHREM) developed by Kizuka et al.,In the present study, grain boundary migration and successive transformation of crystal structure in nanocrystalline ZnO were investigated by TRHREM.Zinc oxide was vacuum-deposited on air-cleaved (001) surfaces of sodium chloride at 200°C. TRHREM was carried out at room temperature using a 200-kV electron microscope (JEOL, JEM2010) equipped with a high sensitive TV camera and a video tape recorder.

Time-resolved high-resolution electron microscopy of structural stability in mgo clusters

Structure and stability of atomic clusters have been studied by time-resolved high-resolution electron microscopy (TRHREM). Typical examples are observations of structural fluctuation in gold (Au) clusters supported on silicon oxide films, graphtized carbon films and magnesium oxide (MgO) films. All the observations have been performed on the clusters consisted of single metal element. Structural stability of ceramics clusters, such as metal-oxide, metal-nitride and metal-carbide clusters, has not been observed by TRHREM although the clusters show anomalous structural and functional properties concerning to solid state physics and materials science.In the present study, the behavior of ceramic, magnesium oxide (MgO) clusters is for the first time observed by TRHREM at 1/60 s time resolution and at atomic resolution down to 0.2 nm.MgO and gold were subsequently deposited on sodium chloride (001) substrates. The specimens, single crystalline MgO films on which Au particles were dispersed were separated in distilled water and observed by using a 200-kV high-resolution electron microscope (JEOL, JEM2010) equipped with a high sensitive TV camera and a video tape recorder system.

Time-resolved high-resolution electron microscopy of grain migration process in MgO films

High-resolution electron microscopy (HREM) has been employed intensively to analyze the atomic structures of grain boundaries and interfaces having two dimensional structures inside polycrystalline and composite materials. Furthermore time-resolved HREM (TRHREM) is required to analyze the behavior of grain boundaries and interfaces at atomic scale. The grain boundary migration, which is a typical grain boundary behavior, is a fundamental process relating to structural stability of polycrystalline materials. The mechanism of the migration has been still unknown.In the present study, the variation of atomic arrangement at the grain boundary migration of a MgO [001]Σ5 boundary was analyzed by TRHREM.Magnesium oxide polycrystalline films were prepared by vacuum-deposition on air-cleaved (001) surfaces of sodium chloride at 300°C. TRHREM was carried out at room temperature using a 200-kV electron microscope (JEOL, JEM2010) equipped with a high sensitive TV camera and a video tape recorder. The spatial resolution of the system was 0.2 nm at 200 kV and the time resolution was 1/60 s. Electron beam density was 30 A/cm2.

Time-resolved high-resolution electron microscopy of nanometer-scale electron beam processing of PbTe films

Various kinds of nanometer scale processings are required to produce advanced materials, for example, nano-structured electric devices. Electron beam processing at nanometer scale using STEM and TEM, such as drilling and line-writing, is recently interested as a most useful method. Details of structural change during the processing should be elucidated at atomic resolution in order to establish the processing. In the present work we have processed lead telluride (PbTe) films with nanometer electron beam in a high-resolution transmission electron microscope and in-situ observed the variation of atomic arrangements during the processing.PbTe of 99.99% was vacuum-deposited on air-cleaved (001) surfaces of sodium chloride at room temperature. Time-resolved high-resolution electron microscopy was carried out at room temperature using a 200-kV electron microscope (JEOL, JEM2010) equipped with a high sensitive TV camera and a video tape recorder. The spatial resolution of thesystem was 0.2 nm at 200 kV and the time resolution was 1/60 s. Electron beam irradiation density was 120 A/cm2 at the processing and the observation.