Practical performance evaluation of a 10k × 10k CCD for electron cryo-microscopy

Electron cryo-microscopy (cryo-EM) images are commonly collected using either charge-coupled devices (CCD) or photographic film. Both film and the current generation of 16 megapixel (4k × 4k) CCD cameras have yielded high-resolution structures. Yet, despite the many advantages of CCD cameras, more than two times as many structures of biological macromolecules have been published in recent years using photographic film. The continued preference to film, especially for subnanometer-resolution structures, may be partially influenced by the finer sampling and larger effective specimen imaging area offered by film. Large format digital cameras may finally allow them to overtake film as the preferred detector for cryo-EM. We have evaluated a 111-megapixel (10k × 10k) CCD camera with a 9 μm pixel size. The spectral signal-to-noise ratios of low dose images of carbon film indicate that this detector is capable of providing signal up to at least 2/5 Nyquist frequency potentially retrievable for 3D reconstructions of biological specimens, resulting in more than double the effective specimen imaging area of existing 4k × 4k CCD cameras. We verified our estimates using frozen-hydrated ε15 bacteriophage as a biological test specimen with previously determined structure, yielding a ～7 ? resolution single particle reconstruction from only 80 CCD frames. Finally, we explored the limits of current CCD technology by comparing the performance of this detector to various CCD cameras used for recording data yielding subnanometer resolution cryo-EM structures submitted to the electron microscopy data bank (http://www.emdatabank.org/).     

Are Private Reserves Effective for Jaguar Conservation?

We present the first study of density and apparent survival for a jaguar (Panthera onca) population in northern Mexico using 13 years of camera trap data from 2000 to 2012. We used the Barker robust design model which combines data from closed sampling periods and resight data between these periods to estimate apparent survival and abundance. We identified 467 jaguar pictures that corresponded to 48 jaguar individuals. We included camera type and field technician as covariates for detection probabilities. We used three covariates to evaluate the effect of reserve on jaguar apparent survival: i) private reserve creation ii) later reserve expansions, and iii) cattle ranches’ conservation activities. We found that the use of digital cameras in addition to film cameras increased detection probability by a factor of 6x compared with the use of only film cameras (p = 0.34 ± 0.05 and p = 0.05 ± 0.02 respectively) in the closed period and more than three times in the open period (R = 0.91 ± 0.08 and R = 0.30 ± 0.13 mixed and film cameras respectively). Our availability estimates showed no temporary emigration and a fidelity probability of 1. Despite an increase of apparent survival probability from 0.47 ± 0.15 to 0.56 ± 0.11 after 2007, no single covariate explained the change in these point estimates. Mean jaguar density was 1.87 ± 0.47 jaguars/100 km². We found that 13 years of jaguar population monitoring with our sampling size were not enough for detecting changes in survival or density. Our results provide a baseline for studies evaluating the effectiveness of protected areas and the inclusion of ranch owners in jaguar conservation programs and long-term population viability.     

Standardizing digital photography: it's not all in the eye of the beholder

Advances in digital photography have made it an efficient and economically appealing alternative to conventional photography. Nevertheless, as objective observers and clinical photographers, we must realize that all digital cameras are not created equal. Different digital cameras frequently used in plastic surgery practices (Olympus 600DL, Olympus 2500, Sony DSC-D700, Nikon Coolpix 950, and Nikon D1) were evaluated, using a subject photographed with each camera in the identical lighting conditions, to determine inherent differences in quality, color, and contrast of the resultant photographs. Three different lighting conditions were examined: single soft-box lighting, dual studio flash boxes, and operating room lighting with on-camera flash. The same digital settings (program mode, ISO camera default setting, high quality setting with JPEG compression) were used. Each camera was digitally color balanced using an 18 percent gray card. Raw and color-balanced images were viewed side-by-side. The macro-image capabilities of each camera were also examined. Conventional 35-mm photographs using a 105 macro-lens on Kodachrome and Ektachrome slide film were obtained for comparison. All of the digital cameras performed with noticeable differences, but they maintained consistency in the three different lighting conditions. Digital photographs differed most greatly with respect to quality and contrast, which was especially obvious once color balancing was performed. Marked differences in quality and ability were observed with respect to macro-image capabilities. Inherent differences in features among digital cameras produce dramatically different photographic results with regard to color, contrast, focus, and overall quality. With the increasing use of digital photography in plastic surgery journals and presentations, it must be recognized that digital cameras do not all display photographs of similar quality, especially when used to evaluate skin appearance. To standardize digital photography, the surgeon must realize that switching digital cameras is akin to switching film types. Standardization of digital photographs should include image resolution between 1.5 and 2.7 million pixels, ISO default setting, color balancing with an 18 percent gray card and software, consistency in focal distance, JPEG compression of medium-to-high quality, and backgrounds of medium blue or 18 percent gray.     

How to convert a traditional electron microscopy laboratory to digital imaging: follow the 'middle road'

Today, electron microscopy (EM) is increasingly confronted by the revolution in image-processing technology provoked by modern computers. Digital cameras are fast replacing film-based cameras in EM, as elsewhere, and the procedures for digital image-archiving, image-analysis, and image publication are rapidly evolving. To take advantage of these advances, we have chosen for the moment a 'middle road', in which film remains our basic recording medium in the electron microscope, but immediately thereafter, all film-based images are converted to digital files for further analysis and processing. The rationale behind this approach is that film still offers far greater sensitivity and resolution (providing an image equivalent to> 10 000 pixels per inch in a 1-s exposure), and film is still far easier to organize and archive than digital images of comparable resolution. However, digital manipulation of EM images has become mandatory. Hence, we explain here, in some detail, how we convert from film to digital.     

Assessing the capabilities of a 4kx4k CCD camera for electron cryo-microscopy at 300kV

CCD cameras have numerous advantages over photographic film for detecting electrons; however the point spread function of these cameras has not been sufficient for single particle data collection to subnanometer resolution with 300kV microscopes. We have adopted spectral signal to noise ratio (SNR) as a parameter for assessing detector quality for single particle imaging. The robustness of this parameter is confirmed under a variety of experimental conditions. Using this parameter, we demonstrate that the SNR of images of either amorphous carbon film or ice embedded virus particles collected on a new commercially available 4kx4k CCD camera are slightly better than photographic film at low spatial frequency (<1/5 Nyquist frequency), and as good as photographic film out to half of the Nyquist frequency. In addition it is slightly easier to visualize ice embedded particles on this CCD camera than on photographic film. Based on this analysis it is realistic to collect images containing subnanometer resolution data (6-9A) using this CCD camera at an effective magnification of approximately 112000x on a 300kV electron microscope.     

Automated image acquisition and processing using a new generation of 4K x 4K CCD cameras for cryo electron microscopic studies of macromolecular assemblies

We have previously reported the development of AutoEM, a software package for semi-automated acquisition of data from a transmission electron microscope. In continuing efforts to improve the speed of structure determination of macromolecular assemblies by electron microscopy, we report here on the performance of a new generation of 4 K CCD cameras for use in cryo electron microscopic applications. We demonstrate that at 120 kV, and at a nominal magnification of 67000 x, power spectra and signal-to-noise ratios for the new 4 K CCD camera are comparable to values obtained for film images scanned using a Zeiss scanner to resolutions as high as approximately 1/6.5A(-1). The specimen area imaged for each exposure on the 4 K CCD is about one-third of the area that can be recorded with a similar exposure on film. The CCD camera also serves the purpose of recording images at low magnification from the center of the hole to measure the thickness of vitrified ice in the hole. The performance of the camera is satisfactory under the low-dose conditions used in cryo electron microscopy, as demonstrated here by the determination of a three-dimensional map at 15 A for the catalytic core of the 1.8 MDa Bacillus stearothermophilus icosahedral pyruvate dehydrogenase complex, and its comparison with the previously reported atomic model for this complex obtained by X-ray crystallography.     

High-throughput film-densitometry: an efficient approach to generate large data sets

A film-handling machine (robot) has been built which can, in conjunction with a commercially available film densitometer, exchange and digitize over 300 electron micrographs per day. Implementation of robotic film handling effectively eliminates the delay and tedium associated with digitizing images when data are initially recorded on photographic film. The modulation transfer function (MTF) of the commercially available densitometer is significantly worse than that of a high-end, scientific microdensitometer. Nevertheless, its signal-to-noise ratio (S/N) is quite excellent, allowing substantial restoration of the output to "near-to-perfect" performance. Due to the large area of the standard electron microscope film that can be digitized by the commercial densitometer (up to 10,000 x 13,680 pixels with an appropriately coded holder), automated film digitization offers a fast and inexpensive alternative to high-end CCD cameras as a means of acquiring large amounts of image data in electron microscopy.     

A scheme for assessing the performance characteristics of small field-of-view gamma cameras

Existing protocols for assessing the performance characteristics of large field-of-view (LFOV) gamma cameras can be inappropriate and require modification for use with small field-of-view (SFOV) gamma camera systems. This communication proposes a generic scheme suitable for evaluating the performance characteristics of SFOV gamma cameras, based on modifications to the standard procedures of NEMA NU1-2007. Key differences in methodology between tests for LFOV and SFOV gamma cameras are highlighted along with the rationale for these changes. It is envisaged that this scheme will provide more appropriate methods for equipment characterisation, ensuring quality and consistency for all SFOV cameras.     

An Assessment of the Effectiveness of High Definition Cameras as Remote Monitoring Tools for Dolphin Ecology Studies

Research involving marine mammals often requires costly field programs. This paper assessed whether the benefits of using cameras outweighs the implications of having personnel performing marine mammal detection in the field. The efficacy of video and still cameras to detect Indo-Pacific bottlenose dolphins (Tursiops aduncus) in the Fremantle Harbour (Western Australia) was evaluated, with consideration on how environmental conditions affect detectability. The cameras were set on a tower in the Fremantle Port channel and videos were perused at 1.75 times the normal speed. Images from the cameras were used to estimate position of dolphins at the water’s surface. Dolphin detections ranged from 5.6 m to 463.3 m for the video camera, and from 10.8 m to 347.8 m for the still camera. Detection range showed to be satisfactory when compared to distances at which dolphins would be detected by field observers. The relative effect of environmental conditions on detectability was considered by fitting a Generalised Estimation Equations (GEEs) model with Beaufort, level of glare and their interactions as predictors and a temporal auto-correlation structure. The best fit model indicated level of glare had an effect, with more intense periods of glare corresponding to lower occurrences of observed dolphins. However this effect was not large (-0.264) and the parameter estimate was associated with a large standard error (0.113). The limited field of view was the main restraint in that cameras can be only applied to detections of animals observed rather than counts of individuals. However, the use of cameras was effective for long term monitoring of occurrence of dolphins, outweighing the costs and reducing the health and safety risks to field personal. This study showed that cameras could be effectively implemented onshore for research such as studying changes in habitat use in response to development and construction activities.     

Temporal characteristics of X-ray emission from X-pinches

The temporal characteristics of X-ray emission from X-pinches have been studied experimentally on the XP facility (with a 100-ns current pulse duration and 470-kA current amplitude) at Cornell University. The experiments were performed with X-pinches made of Al, Ti, Mo, and W wires. Radiation in the photon energy range 1–10 keV was recorded using diamond and silicon photodiodes with a subnanosecond time resolution and X-ray streak cameras with a picosecond time resolution. It is shown that, for high-Z elements, the duration of the X-ray pulse in the short-wavelength part of the spectral range under study does not exceed 5–10 ps.     

A place of digital X-ray fluorography in the detection of lung diseases in the public health facilities of the Russian Federation

The paper shows the authors' opinion as to the use of digital X-ray fluorography (DXF) to detect pathology of the lung, primarily its tuberculosis and cancer from organizational-and-methodological and purely diagnostic standpoints. Based on a great body of data pooling 21,295 studies of patients, which comprised two (screening and routine clinical) groups, they authors state their views on the place and role of lung DXF in the work of public health facilities in the Russian Federation (RF). The objectivity of the authors' opinion is confirmed by the different nature of a lung abnormality detected by DXF at both the municipal and regional levels of the RF public health system and by a comparative analysis of the findings with those of film fluorography, routine X-ray study, linear tomography, X-ray computed tomography (XCT), and surgical evidence. The study has allowed the authors to express their opinion by recommending DXF as a method for primary diagnosis of lung diseases instead of film fluorography and routine X-ray study and to appreciate it in screenings. The authors' data indicate that XCT in its simplest variant rather than linear tomography should become the optimum method that specifies the diagnosis of lung diseases detected by DXT.     

多层螺旋CT检查与腹部X线平片对急性肠梗阻的诊断价值比较研究

目的:研究比较多层螺旋CT(MSCT)检查与腹部X线平片对急性肠梗阻(AIO)的诊断价值。方法:选择2016年1月到2018年4月间在蚌埠医学院附属阜阳医院接受手术治疗的200例AIO患者作为研究对象,对所有患者先常规予以腹部X线平片诊断,12h后再通过MSCT为患者实施诊断,对比两种方法的诊断结果、诊断体验效果以及漏诊率和误诊率。结果:MSCT的肠梗阻检出率为94.50%,明显较腹部X线平片的69.00%更高(P0.05)。MSCT所诊断的肠梗阻中,梗阻类型为绞窄型及梗阻病因为肠肿瘤者均占100.00%,较腹部X线平片的36.21%和54.26%明显更高(P0.05)。MSCT的诊断舒适度评分、图像清晰度评分较腹部X线平片明显更高,而操作复杂度评分较腹部X线平片明显更低(P0.05)。MSCT的漏诊率、误诊率分别为4.00%、1.50%,较腹部X线平片的22.00%、9.00%明显降低(P0.05)。结论:对于AIO患者,MSCT较腹部X线平片具有更高的诊断价值,诊断体验效果更好,漏诊率和误诊率偏低。     

The use of Polaroid Land film in radioautography

It is shown that Polaroid film may be used in place of X-ray film in many experiments employing radioautographic techniques. In ¹⁴C experiments, the sensitivity of PolaPan Type 57 film is at least comparable to that of X-ray film, while radioactive iodine results in a slight increase in sensitivity of the Polaroid film over X-ray film. Darkroom facilities and time-consuming developing procedures are not necessary when Polaroid film is used. The background exposure usually acquired by X-ray film is nonexistent with Polaroid film. Under certain circumstances the differentiation of isotopes is possible in double-isotope experiments.     

Accuracy assessment and control point configuration when using the DLT for photogrammetry.

The direct linear transformation (DLT) is a common technique used to calibrate cameras and subsequently reconstruct points filmed with two or more cameras in a three-dimensional object space. The assessment of the accuracy of this technique, and of the influence of the distribution of control points on accuracy were examined. It was concluded that to obtain a true estimation of reconstruction accuracy, an independent assessment criterion is required, and that the use of control points distributed around the outside, rather than within the space to be calibrated, is preferred.     

Fabrication of carbon films with ∼500 nm holes for cryo-EM with a direct detector device

Single particle electron cryomicroscopy (cryo-EM) is often performed using EM grids coated with a perforated or holey layer of amorphous carbon. Regular arrays of holes enable efficient cryo-EM data collection and several methods for the production of micropatterned holey-carbon film coated grids have been described. However, a new generation of direct detector device (DDD) electron microscope cameras can benefit from hole diameters that are smaller than currently available. Here we extend a previously proposed method involving soft lithography with a poly(dimethylsiloxane) (PDMS) stamp for the production of holey-carbon film coated EM grids. By incorporating electron-beam (e-beam) lithography and modifying the procedure, we are able to produce low-cost high-quality holey-carbon film coated EM grids with ∼500 nm holes spaced 4 μm apart centre-to-centre. We demonstrate that these grids can be used for cryo-EM. Furthermore, we show that by applying image shifts to obtain movies of the carbon regions beside the holes after imaging the holes, the contrast transfer function (CTF) parameters needed for calculation of high-resolution cryo-EM maps with a DDD can be obtained efficiently.     

Three-dimensional X-ray observation of atmospheric biological samples by linear-array scanning-electron generation X-ray microscope system

Recently, we developed a soft X-ray microscope called the scanning-electron generation X-ray microscope (SGXM), which consists of a simple X-ray detection system that detects X-rays emitted from the interaction between a scanning electron beam (EB) and the thin film of the sample mount. We present herein a three-dimensional (3D) X-ray detection system that is based on the SGXM technology and designed for studying atmospheric biological samples. This 3D X-ray detection system contains a linear X-ray photodiode (PD) array. The specimens are placed under a CuZn-coated Si₃N₄ thin film, which is attached to an atmospheric sample holder. Multiple tilt X-ray images of the samples are detected simultaneously by the linear array of X-ray PDs, and the 3D structure is calculated by a new 3D reconstruction method that uses a simulated-annealing algorithm. The resulting 3D models clearly reveal the inner structure of the bacterium. In addition, the proposed method can easily be used for diverse samples in a broad range of scientific fields.     

Photography in limnology: documentation of lake color using a CCD camera

During a survey of Norwegian lakes, photographic records were made of lake color as reflected by a white Secchi disk positioned at half of the depth of extinction. The pictorial distinction between different lakes is documented in this article. The pictures recorded can readily be transferred to a color model [e.g., Commission Internationale de L’Éclairage (CIE)-Lab]; from there, numerical color parameter values such as hue (h*, the quality of color) and chroma (C*, the intensity of color) may be assigned to each record. There are, however, limitations and obstacles connected with the transformation of color values recorded by a CCD camera (or film digitizers) to absolute numerical values comparable between different cameras and systems. A single CCD camera may be useful for documenting lake color, but there are technical limitations restricting its use as a scientific instrument for quantitative purposes.     

Experience with computerized technologies in the diagnosis of respiratory diseases in children

The paper summarizes the experience gained in using an automatic X-ray study system (AXRSS) for 5 years. AXRSS is successful in tackling the task of having a display image of the quality that allows one to make X-ray diagnosis with the same confidence as that of an X-ray film.     

Feasibility of using orthogonal fluoroscopic images to measure in vivo joint kinematics

Accurately determining in vivo knee kinematics is still a challenge in biomedical engineering. This paper presents an imaging technique using two orthogonal images to measure 6 degree-of-freedom (DOF) knee kinematics during weight-bearing flexion. Using this technique, orthogonal images of the knee were captured using a 3-D fluoroscope at different flexion angles during weight-bearing flexion. The two orthogonal images uniquely characterized the knee position at the specific flexion angle. A virtual fluoroscope was then created in solid modeling software and was used to reproduce the relative positions of the orthogonal images and X-ray sources of the 3-D fluoroscope during the actual imaging procedure. Two virtual cameras in the software were used to represent the X-ray sources. The 3-D computer model of the knee was then introduced into the virtual fluoroscope and was projected onto the orthogonal images by the two virtual cameras. By matching the projections of the knee model to the orthogonal images of the knee obtained during weight-bearing flexion, the knee kinematics in 6 DOF were determined. Using regularly shaped objects with known positions and orientations, this technique was shown to have an accuracy of 0.1 mm and 0.1 deg in determining the positions and orientations of the objects, respectively.