Slow-scan Charge-coupled Device Camera Research Articles

Molecular Details of Bax Activation, Oligomerization, and Membrane Insertion

Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices alpha5 and alpha6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.

Effect of Molecules Secreted by Lactobacillus acidophilus Strain La-5 on Escherichia coli O157:H7 Colonization

The probiotic bacterium Lactobacillus acidophilus strain La-5 is a gut-colonizing microorganism that, when established, becomes an important part of the gastrointestinal (GI) tract microbiota. It has been shown to be effective against enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection. We have previously shown that molecules released by probiotic strain La-5 influence the transcription of EHEC genes involved in colonization and quorum sensing. In this work, we report on the ability of these molecules to prevent the adherence of EHEC to epithelial cells and on its capacity to concentrate F-actin at adhesion sites. With a fluorescein-labeled phallotoxin, it was shown that La-5 cell-free spent medium (CFSM) fractions remarkably reduced attaching and effacing lesions in HeLa cells. We also observed a significant inhibition of bacterial adhesion to Hep-2 cells when they were treated with the same La-5 CFSM fractions. In order to observe if La-5 CFSM fractions exhibited the same effect in vivo, we studied the ability of luminescent EHEC constructs (LEE1::luxCDABE) to adhere to intestinal epithelial cells of specific-pathogen-free ICR mice following intragastric inoculation. Colonization of the GI tract by luminescent EHEC O157:H7 was monitored in real time with a slow-scan charge-coupled device camera. At the same time, fecal shedding of EHEC was studied. Following oral gavage of the La-5 active fraction, we observed a reduced amount of bioluminescence signal along with a decrease in fecal shedding by mice, indicating an effect on the ability of the organism to colonize the GI tract. Our results confirm past evidence of the possibility of blocking or interfering with EHEC's virulence by active molecules contained in the probiotic CFSM and identify novel therapeutic alternatives to antibiotic treatments in the fight against food-borne pathogens.

Atomic structure of YB56 studied by digital high-resolution electron microscopy and electron diffraction

Atomic positions for Y atoms were determined by using high-resolution electron microscopy and electron diffraction. A slow-scan charge-coupled device camera which had high linearity and electron sensitivity was used to record high-resolution images and electron diffraction patterns digitally. Crystallographic image processing was applied for image analysis, which provided more accurate, averaged Y atom positions. In addition, atomic disordering positions in YB56 were detected from the differential images between observed and simulated images based on x-ray data, which were B24 clusters around the Y-holes. The present work indicates that the structure analysis combined with digital high-resolution electron microscopy, electron diffraction, and differential images is useful for the evaluation of atomic positions and disordering in the boron-based crystals.

Atomic disordering in YB56detected by high-resolution electron microscopy with residual indices

Abstract Disordering of both yttrium and boron atoms in YB56 was observed, in three dimensions, from difference images of high-resolution electron microscopy (HREM) and simulated images from an X-ray determined structural model. Averaged HREM images recorded using a slow-scan charge-coupled device camera from thin regions (less than 5 nm) along the [100], [110] and [111] directions of the YB56 were compared with calculated images based on the X-ray data. A residual index R HREM was used for image analysis to determine the crystal thickness and defocus value of the observed images. Based on the experimental conditions which minimized the R HREM values, difference images between observed and simulated images were calculated. These difference images observed along the three directions of the YB56 showed the atomic disorder, which are yttrium positions and boron clusters around the yttrium atoms. This approach is useful for the evaluation of disordered atomic positions in the crystal.

Atomic disordering in YB56 detected by high-resolution electron microscopy with residual indices

Abstract Disordering of both yttrium and boron atoms in YB56 was observed, in three dimensions, from difference images of high-resolution electron microscopy (HREM) and simulated images from an X-ray determined structural model. Averaged HREM images recorded using a slow-scan charge-coupled device camera from thin regions (less than 5 nm) along the [100], [110] and [111] directions of the YB56 were compared with calculated images based on the X-ray data. A residual index R HREM was used for image analysis to determine the crystal thickness and defocus value of the observed images. Based on the experimental conditions which minimized the R HREM values, difference images between observed and simulated images were calculated. These difference images observed along the three directions of the YB56 showed the atomic disorder, which are yttrium positions and boron clusters around the yttrium atoms. This approach is useful for the evaluation of disordered atomic positions in the crystal.

CCD-camera based capillary chemiluminescent detection of retinol binding protein

A chemiluminescence (CL) assay for retinol-binding protein (RBP) was designed and optimized using a charge coupled device (CCD)-camera based detection system. A sandwich ELISA was designed based on the anti-RBP antibodies immobilized in glass capillaries pre-treated with silica sol. The immobilization was predominantly by physisorption of the protein on the silica surface. The RBP bound to the anti-RBP antibodies was detected by using an anti-RBP-horseradish peroxidase (HRP) conjugate. The reaction of the HRP with hydrogen peroxide and luminol and 4-iodophenol generated the CL. The CL emitted from the glass capillaries was detected by a cooled slow scan CCD camera at an optimized exposure time. The approximately linear range of RBP determination was between 11 pg ml −1 and 11 ng ml −1 with a coefficient of variation of 5–11% ( n=6) over this range.

Detection of bioluminescence from individual bacterial cells: a comparison of two different low‐light imaging systems

Detection of very low light levels arising from individual cells of the naturally bioluminescent bacterium Vibrio fischeri as well as from a luminescence-marked Pseudomonas putida strain was achieved by the aid of two different camera systems. Using a liquid nitrogen-cooled slow-scan CCD (charge-coupled device) camera we were able to detect single-cell bioluminescence within 1 min, and the pictures obtained were of good resolution. In contrast, employing a photon-counting video camera we were able to detect bioluminescent cells within 10 seconds, but at the expense of spatial resolution. This study demonstrates the feasibility of microscopic single cell analysis employing bioluminescence as reporter system. © 1997 John Wiley & Sons, Ltd.

High-resolution high-speed synchronous epifluorescence imaging of cardiac activation

An optical imaging technique with high spatial and temporal resolution was developed to record fractional changes in laser-induced epifluorescence associated with the cardiac transmembrane potential during and after the application of monophasic point stimuli. The technique takes advantage of the repeatability of the recorded events, and uses a synchronized laser strobing mechanism to overcome the speed limitation inherent to slow-scan charge-coupled device cameras, and achieves an effective frame rate of 500 frames/s at a spatial resolution of 100×100 pixels in a single frame with a pixel resolution of 75 μm. The signal-to-noise ratio can be improved with boxcar averaging. Patterns of virtual cathode and anode with distinctive regions of simultaneous depolarization and hyperpolarization during stimulation are demonstrated with stimuli applied to the resting myocardium of an isolated rabbit heart. The technique described in this article provides a powerful tool for investigating repeatable dynamics in the function of electrically active tissue.

Single particle tracking of cell-surface HLA-DR molecules using R-phycoerythrin labeled monoclonal antibodies and fluorescence digital imaging.

The mobility of cell surface MHC molecules and their ability to form dynamic associations may be related to the physiological status of the cell and to the potential to bind effector T lymphocytes. To investigate these properties, we have prepared HLA DR specific monoclonal antibodies coupled in a 1:1 mole ratio to the fluorescent phycobiliprotein, R-phycoerythrin (PE). We show that these small particles can be sequentially imaged using a cooled slow-scan charge coupled device camera and hence can be used for single particle tracking experiments. We have applied this technique to investigate the movements of HLA DR molecules on fibroblasts transfected with human DR alpha and DR beta genes. PE-IgG was bound to the transfected fibroblasts and particle tracks were obtained by sequential imaging over a period of typically 30 minutes. Analysis of particle tracks revealed the presence of directed motion and domain-limited diffusion in addition to random diffusion. The contributions of these three types of motion showed cell to cell variability. Velocities of directed motion were of the order of 2 nm second-1 whilst domain diameters were in the range 200-800 nm. Diffusion coefficients for random diffusion were in the range 1 x 10(-13)-5 x 10(-12) cm2 second-1. The higher mobilities were observed for the lower intensity fluorescent spots, which possibly correspond to images of single particles. Much lower mobility was observed with a cell where the spot intensities were approximately double that of the lower intensity spots. These spots could be images of double particles implying the association of at least two HLA DR alpha beta dimers. These data are relevant to the study of MHC class II cell surface redistribution and antigen presentation in specific immunity.

Performance of a slow-scan CCD camera for macromolecular imaging in a 400 kV electron cryomicroscope

The feasibility and limitations of a 1024 × 1024 slow-scan charge-coupled device (CCD) camera were evaluated for imaging in a 400kV electron cryomicroscope. Catalase crystals and amorphous carbon film were used as test specimens. Using catalase crystals, it was found that the finite (24 μm) pixel size of the slow-scan CCD camera governs the ultimate resolution in the acquired images. For instance, spot-scan images of ice-embedded catalase crystals showed resolutions of 8Å and 4Å at effective magnifications of 67,000 × and 132,000 ×, respectively. Using an amorphous carbon film, the damping effect of the modulation transfer function (MTF) of the slow-scan CCD camera on the specimen's Fourier spectrum relative to that of the photographic film was evaluated. The MTF of the slow-scan CCD camera fell off more rapidly compared to that of the photographic film and reached the value of 0.2 at the Nyquist frequency. Despite this attenuation, the signal-to-noise ratio of the CCD data, as determined from reflections of negatively-stained catalase crystals, was found to decrease to ~50% of that of photographic film data. The phases computed from images of the same negatively-stained catalase crystals recorded consecutively on both the slow-scan CCD camera and photographic film were found to be comparable to each other within 12 °. Ways of minimizing the effect of the MTF of the slow-scan CCD camera on the acquired images are also presented.

A low-energy electron diffraction data acquisition system for very low electron doses based upon a slow scan charge coupled device camera

We have developed a video low-energy electron diffraction (LEED) system on the basis of a slow scan charge coupled device (CCD) camera which is capable of collecting LEED IV data at very low electron doses quickly and therefore enables us to study extremely beam sensitive surface structures which have not been accessible to LEED IV analysis before. The slow scan CCD camera allows separating the relatively short data acquisition process from the more lengthy digitizing, storage, and data analysis processes. Typical total effective exposure times can therefore be reduced to about 200–300 s (1 s per energy point) at a primary beam current of 100 nA which corresponds to a total dose of about 12 e per adsorbate particle; further decrease is possible. The total measurement time for collecting a complete set of LEED images is of the order of 30–40 min which assures the exclusion of contamination effects, even for sensitive layers. The IV curves are then extracted from the digitally stored images off-line which allows collecting the intensities of all visible spots simultaneously with a high reliability in tracing beams, even for very dense LEED patterns.

Application of a slow-scan CCD camera in protein electron crystallography at 400 KV

In protein electron crystallography, high resolution amplitudes and phases are required for a 3D reconstruction of a protein in which one can trace the Cα-backbone. Recording of these amplitudes and phases has so far been done on photographic film. In principle they can be obtained with higher fidelity using an electronic device, such as a slow-scan charge-coupled device (CCD) camera. We use a 1024 × 1024 Gatan CCD camera (model 679) attached to a JEOL 4000EX intermediate voltage electron cryo-microscope. The camera has a 20-30 μm thin P43 phosphor scintillator and an extra set of fiber optics, that reduces the number of x-ray pixels in acquired frames. In this abstract, we will focus on several issues related to the CCD camera in obtaining high resolution diffraction patterns and images from thin protein crystals.To obtain electron diffraction patterns with sufficient statistical definition, one needs to use an adequate electron dose. However, at exposures well above 0.25 e/Å2, the pattern will suffer from blooming due to the central beam.

Three-dimensional structure of lipid vesicles embedded in vitreous ice and investigated by automated electron tomography

Automated electron tomography is shown to be a suitable means to visualize the shape of phospholipid vesicles embedded in vitrified ice. With a slow-scan charge-coupled device camera as a recording device, the cumulative electron dose needed to record a data set of 60 projections at a magnification of 20,000X can be kept as low as 15 e-/A2 (or 1500 electrons/nm2). The membrane of the three-dimensionally reconstructed vesicles is clearly visible in two-dimensional sections through the three-dimensionally reconstructed volume. Some edges indicating a polygonal shape of the vesicles, frozen from the gel phase, are also clearly recognized. Because of the presently limited tilt angle range (+/- 60 degrees), the upper and lower "caps" of the vesicles (representing about 35% of the surface of the ellipsoidal particles) remain invisible in the three-dimensional reconstruction.

Applications of a Slow-Scan CCD Camera in Protein Electron Crystallography

A Gatan 1024 × 1024 slow-scan charge-coupled device (CCD) camera has been interfaced to a JEOL4000EX electron cryomicroscope and explored for its usefulness in the electron crystallographic analysis of thin, glucose-embedded crystals of crotoxin complex kept at -125°C. We show that the camera allows for an on-line assessment of the crystals' crystallinity, flatness, and thickness. Intensities obtained from electron diffraction patterns acquired with the camera have been statistically analyzed and were found to be consistent with theoretically expected values. A quantitative analysis of the diffraction intensity as function of the accumulated electron dose suggests the possibility of recording up to 250 diffraction patterns with 3.5 Å resolution from a single crotoxin complex crystal 128 Å thick. Tilt series of 125 electron diffraction patterns with 3.5 Å data acquired from a single crystal are shown to be practically feasible. The current study demonstrates for the first time the effectiveness of using a slow-scan CCD camera for electron diffraction data collection from thin protein crystals at near atomic resolution.

MEASUREMENT OF THE RATE OF UPTAKE AND SUBCELLULAR LOCALIZATION OF PORPHYRINS IN CELLS USING FLUORESCENCE DIGITAL IMAGING MICROSCOPY

A fluorescence imaging system incorporating a cooled slow-scan charge-coupled device camera was used to study the rate of uptake and subcellular localization of prophyrins in living cells. Measurements were carried out on human dermal fibroblasts (D532) using two different prophyrins meso-tetra(4-N-methylpyridyl)porphine (TMPP) and meso-tetra(4-N-hexylpyridyl)porphine (THPP). It was observed that TMPP was rapidly taken up by cells and principally located in the nucleus. The THPP, on the other hand, internalized more slowly and exhibited a particulate distribution in the cytoplasm.

High-sensitivity lens-coupled slow-scan CCD camera for transmission electron microscopy

A lens-coupled slow-scan CCD camera has been built for transmission electron microscopy (TEM) applications. In this design, a leaded glass window, which is coated with a 20 μm layer of red P20 phosphor, is mounted on the bottom of the microscope. A lens assembly and mirror prism, located outside the microscope vacuum below the leaded glass, relays the image onto a back-thinned 1k×1k charge-coupled device (CCD) detector. This CCD is electronically cooled to below -30°C during operation. It is found that X-ray irradiation, generally found to be annoying in fiber-optically coupled CCD cameras, is completely eliminated by this configuration. The collection efficiency of this system, although not as high as some of the fiber-optically coupled CCD cameras, is high enough to achieve single-electron sensitivity under a high-gain mode.

Methods to measure properties of slow-scan CCD cameras for electron detection

New ways to measure detection properties of slow-scan charge-coupled device (CCD) cameras suitable for electron microscopy, mainly based on the statistics of single-electron events, are discussed. The experiments concentrate on the newly introduced Gatan 679 slow-scan CCD camera. It has been established that for this instrument (if equipped with a thin YAG scintillator) 20% of the true intensity is recorded at the highest detectable spatial frequency (Nyquist frequency), limited mostly by the modulation transfer function of the YAG scintillator. The detection quantum efficiency is 0.45 for single 100-kV electrons and 0.15 for single 400-kV electrons, and is approaching unity for intensities higher than ten electrons. Furthermore, nonlinearity of the response is smaller than deviations in the image intensity due to shot noise. Examples are presented illustrating the detection properties of slow-scan CCD cameras for electron imaging, which also include high dynamic range and negligible geometric distortion.

Application of slow-scan CCD cameras in routine biological TEM

Slow scan CCD (charge-coupled device) cameras (SSCs) are based on image detection technology introduced in the seventies and first used in astronomy in the eighties. When they are properly configured for the detection of medium energy electrons rather than photons, they are able to capture images whose quality exceeds the quality of images recorded on photographic film. Here we discuss aspects of their operation pertinent to routine biological TEM.Compared to the TV-rate CCD sensors that have become ubiquitous in miniature camcorders, scientific (astronomy-grade) CCDs have several unique characteristics: 1) their pixels are typically 20-27 μm in size rather than the 6 μm typical of TV rate CCDs, 2) their read-out amplifiers are optimized for low noise at read out rates of 1/10 to 1/100 of TV rate, 3) they do not have a separate frame-transfer buffer for CCD read-out, and 4) they are available in large total sizes (up to 2048 × 2048 pixels).