EPiK-a Workflow for Electron Tomography in Kepler

CT perfusion (CTP) is obtained cerebrovascular circulation image for assessment of stroke patients; however, at the expense of increased radiation dose by dynamic scan. Iterative reconstruction (IR) method is possible to decrease image noise, it has the potential to reduce radiation dose. The purpose of this study is to assess the visual effect of IR method by using a digital perfusion phantom. The digital perfusion phantom was created by reconstructed filtered back projection (FBP) method and IR method CT images that had five exposure doses. Various exposure dose cerebral blood flow (CBF) images were derived from deconvolution algorithm. Contrast-to-noise ratio (CNR) and visual assessment were compared among the various exposure dose and each reconstructions. Result of low exposure dose with IR method showed, compared with FBP method, high CNR in severe ischemic area, and visual assessment was significantly improvement. IR method is useful for improving image quality of low-dose CTP.

Sampling limits for electron tomography with sparsity-exploiting reconstructions

Knowledge of three-dimensional (3D) structures of each individual particles of asymmetric and flexible proteins is essential in understanding those proteins’ functions; but their structures are difficult to determine. Electron tomography (ET) provides a tool for imaging a single and unique biological object from a series of tilted angles, but it is challenging to image a single protein for three-dimensional (3D) reconstruction due to the imperfect mechanical control capability of the specimen goniometer under both a medium to high magnification (approximately 50,000–160,000×) and an optimized beam coherence condition. Here, we report a fully mechanical control method for automating ET data acquisition without using beam tilt/shift processes. This method could reduce the accumulation of beam tilt/shift that used to compensate the error from the mechanical control, but downgraded the beam coherence. Our method was developed by minimizing the error of the target object center during the tilting process through a closed-loop proportional-integral (PI) control algorithm. The validations by both negative staining (NS) and cryo-electron microscopy (cryo-EM) suggest that this method has a comparable capability to other ET methods in tracking target proteins while maintaining optimized beam coherence conditions for imaging.

Electron tomography (ET) is an outstanding technique to access information about nanoparticles whose properties are critically dependent on their 3D shape and structure. A particularly relevant application of ET concerns plasmonic chiral nanoparticles, which exhibit strong correlation between optical activity and asymmetric morphology. Thus, it is crucial to go beyond qualitative visual inspection of ET reconstructions and extract quantitative shape or chirality descriptors. However, the time‐consuming nature of conventional ET, where a series of 2D projection images is used for one 3D reconstruction, makes it challenging to collect statistically representative data. A recently proposed semi‐automated approach for the acquisition of the required tilt series can reduce the data collection time from typically ≈ 45 minutes–1 hour to less than 10 minutes. What remains unclear is how this method influences the quality of the reconstruction and the accuracy of the samples’ quantitative descriptors. Here, two sets of chiral gold nanoparticles with wrinkled or twisted morphological chirality are used. It is demonstrated that the fast tilt series acquisitions yield results that are consistent with conventional ET, provided a standardized data processing protocol is used. Finally, a practical example of how fast ET can be used to obtain more statistically relevant measurements is presented.

Electron tomography (ET) is a method for three-dimensional (3-D) reconstruction of single, transparent objects from a series of images (i.e., a tilt series) recorded with a transmission electron microscope (EM) (Fig. 4.1). The method is related to the procedures used in medical tomography. The 3-D reconstructions are usually computed from the digitized tilt series after a radial weighting scheme has been applied to the Fourier-transformed data. The ET method can be used to reconstruct in 3-D any object that is transparent enough for projection imaging with the transmission EM. This means that specimens of biological origin are usually available for ET 3-D reconstruction, whereas, e.g., colloidal gold particles are not. An ET-calculated 3-D map could be based on projections from objects that have been visualized by EM in several different ways, i.e., from stained or unstained objects, objects visualized at different energy loss levels or objects embedded in different media. The combined analysis of the 3-D structure, imaged in different ways, could thus become very informative. The general applicability also means that it is not restricted to symmetrical or regularly arranged objects, nor to objects with preferred orientations on a support grid. In its present state, the ET method allows reproducible 3-D reconstructions of single molecular objects, with a resolution in the range of 5 nm, of complex cellular specimens. For isolated objects, free of interfering cellular substances, a somewhat higher resolution can be achieved. The ET method covers the intermediate resolution range where there is no other physical technique available to analyze single molecular complexes.

High-Throughput Immunofluorescence and Electron Tomography to Characterize Centrosomal Aberrations in Plasma Cell Neoplasia

This paper reports the preliminary results of a new in-situ three-dimensional (3D) imaging system for observing plastic deformation behavior in a transmission electron microscope (TEM) as a directly relevant development of the recently reported straining-and-tomography holder [Sato K et al. (2015) Development of a novel straining holder for transmission electron microscopy compatible with single tilt-axis electron tomography. Microsc. 64: 369-375]. We designed an integrated system using the holder and newly developed straining and image-acquisition software and then developed an experimental procedure for in-situ straining and time-resolved electron tomography (ET) data acquisition. The software for image acquisition and 3D visualization was developed based on the commercially available ET software TEMographyTM. We achieved time-resolved 3D visualization of nanometer-scale plastic deformation behavior in a Pb-Sn alloy sample, thus demonstrating the capability of this system for potential applications in materials science.

Chirality in gold nanostructures offers an exciting opportunity to tune their differential optical response to left- and right-handed circularly polarized light, as well as their interactions with biomolecules and living matter. However, tuning and understanding such interactions demands quantification of the structural features that are responsible for the chiral behavior. Electron tomography (ET) enables structural characterization at the single-particle level and has been used to quantify the helicity of complex chiral nanorods. However, the technique is time-consuming and consequently lacks statistical value. To address this issue, we introduce herein a high-throughput methodology that combines images acquired by secondary electron-based electron beam-induced current (SEEBIC) with quantitative image analysis. As a result, the geometric chirality of hundreds of nanoparticles can be quantified in less than 1 h. When combining the drastic gain in data collection efficiency of SEEBIC with a limited number of ET data sets, a better understanding of how the chiral structure of individual chiral nanoparticles translates into the ensemble chiroptical response can be reached.

BackgroundPositron Emission Tomography (PET), Computed Tomography (CT), PET/CT and Single Photon Emission Tomography (SPECT) are non-invasive imaging tools used for creating two dimensional (2D) cross section images of three dimensional (3D) objects. PET and SPECT have the potential of providing functional or biochemical information by measuring distribution and kinetics of radiolabelled molecules, whereas CT visualizes X-ray density in tissues in the body. PET/CT provides fused images representing both functional and anatomical information with better precision in localization than PET alone.Images generated by these types of techniques are generally noisy, thereby impairing the imaging potential and affecting the precision in quantitative values derived from the images. It is crucial to explore and understand the properties of noise in these imaging techniques. Here we used autocorrelation function (ACF) specifically to describe noise correlation and its non-isotropic behaviour in experimentally generated images of PET, CT, PET/CT and SPECT.MethodsExperiments were performed using phantoms with different shapes. In PET and PET/CT studies, data were acquired in 2D acquisition mode and reconstructed by both analytical filter back projection (FBP) and iterative, ordered subsets expectation maximisation (OSEM) methods. In the PET/CT studies, different magnitudes of X-ray dose in the transmission were employed by using different mA settings for the X-ray tube. In the CT studies, data were acquired using different slice thickness with and without applied dose reduction function and the images were reconstructed by FBP. SPECT studies were performed in 2D, reconstructed using FBP and OSEM, using post 3D filtering. ACF images were generated from the primary images, and profiles across the ACF images were used to describe the noise correlation in different directions. The variance of noise across the images was visualised as images and with profiles across these images.ResultsThe most important finding was that the pattern of noise correlation is rotation symmetric or isotropic, independent of object shape in PET and PET/CT images reconstructed using the iterative method. This is, however, not the case in FBP images when the shape of phantom is not circular. Also CT images reconstructed using FBP show the same non-isotropic pattern independent of slice thickness and utilization of care dose function. SPECT images show an isotropic correlation of the noise independent of object shape or applied reconstruction algorithm. Noise in PET/CT images was identical independent of the applied X-ray dose in the transmission part (CT), indicating that the noise from transmission with the applied doses does not propagate into the PET images showing that the noise from the emission part is dominant. The results indicate that in human studies it is possible to utilize a low dose in transmission part while maintaining the noise behaviour and the quality of the images.ConclusionThe combined effect of noise correlation for asymmetric objects and a varying noise variance across the image field significantly complicates the interpretation of the images when statistical methods are used, such as with statistical estimates of precision in average values, use of statistical parametric mapping methods and principal component analysis. Hence it is recommended that iterative reconstruction methods are used for such applications. However, it is possible to calculate the noise analytically in images reconstructed by FBP, while it is not possible to do the same calculation in images reconstructed by iterative methods. Therefore for performing statistical methods of analysis which depend on knowing the noise, FBP would be preferred.

Reprint of “Fiducial-less alignment of cryo-sections” [J. Struct. Biol. 159 (2007) 413–423

3D quantitative characterization of metal catalysts supported on heavy oxides by High Angle Annular Dark Field (HAADF) STEM electron tomography (ET) is a very challenging task. Noble metal nanoparticles (Au, Ru) supported on ceria or ceria mixed oxides illustrates the case. The difference, very small in some cases, between the atomic number (Z) of metal and support (Z Ru =44, Z Au =79, Z Ce =58) complicates the discrimination of the small particles. This makes that each step, acquisition, alignment, reconstruction and segmentation, has to be carefully optimized not only to improve the visualization of the nanoparticles but also to extract relevant quantitative results, e.g . metal loading, specific surface area, or particle size distribution. In this work, we have combined advanced image processing algorithms, based on undecimated wavelets transform (UWT) [1], to improve the contrast and denoising the tilt series projections, with new reconstruction algorithms, based on compressed sensing (CS) [2], to study a series of catalysts with a high potential in different processes related to the production of hydrogen, as CeO 2 Nanorods, Au/CeO 2 Nanocubes or Ru/Ce 2 Z 2 O 8 . The results obtained by ET have been compared to those determined by macroscopic characterization techniques as Inductively Coupled Plasma (ICP) or Brunauer, Emmett and Teller (BET) isotherm method. HAADF‐STEM ET has been carried out using a FEI TITAN 3 THEMIS 60‐300 operated at 200kV recently installed at Cadiz University. Data collections were obtained by tilting the specimen about a single axis perpendicular to the electron beam. Series of projections were acquired between ‐70º and +70° either every 2° or 5º. The images series were aligned using Inspect3D and TomoJ and reconstructed using the ASTRA Toolbox implemented in Matlab [3]. In the particular case of CS, Total Variation Minimization (TVM) was carried by using the TVAL3 solver [2]. Figure 1 shows the 3D‐rendered voxel of a catalyst consisting of Au nanoparticles supported on CeO 2 Nanocubes after reconstructing the raw tilt series by SIRT (Figure 1a) and TVM (Figure 1b) and after denoising the tilt series projections by UWT and reconstructed by SIRT (Figure 1c) and TVM (Figure1d). Note how in the case of UWT‐TVM reconstruction an important improvement in terms of morphology of the support and visualization of nanoparticles is obtained. The 3D rendered surface of a similar sample and the 3D quantifications of relevant properties as metal loading, specific surface area and average particle size are shown in Figure 2. It is important to point out how the values are quite similar to those determined by macroscopic characterization techniques. These results indicate that this combination of techniques allows determining nanostructural features representative of the catalysts at macroscopic level.

The objective of this study was to evaluate the radiation dose reduction potential of a novel image-based denoising technique in pediatric abdominopelvic and chest CT examinations and compare it with a commercial iterative reconstruction method. Data were retrospectively collected from 50 (25 abdominopelvic and 25 chest) clinically indicated pediatric CT examinations. For each examination, a validated noise-insertion tool was used to simulate half-dose data, which were reconstructed using filtered back-projection (FBP) and sinogram-affirmed iterative reconstruction (SAFIRE) methods. A newly developed denoising technique, adaptive nonlocal means (aNLM), was also applied. For each of the 50 patients, three pediatric radiologists evaluated four datasets: full dose plus FBP, half dose plus FBP, half dose plus SAFIRE, and half dose plus aNLM. For each examination, the order of preference for the four datasets was ranked. The organ-specific diagnosis and diagnostic confidence for five primary organs were recorded. The mean (± SD) volume CT dose index for the full-dose scan was 5.3 ± 2.1 mGy for abdominopelvic examinations and 2.4 ± 1.1 mGy for chest examinations. For abdominopelvic examinations, there was no statistically significant difference between the half dose plus aNLM dataset and the full dose plus FBP dataset (3.6 ± 1.0 vs 3.6 ± 0.9, respectively; p = 0.52), and aNLM performed better than SAFIRE. For chest examinations, there was no statistically significant difference between the half dose plus SAFIRE and the full dose plus FBP (4.1 ± 0.6 vs 4.2 ± 0.6, respectively; p = 0.67), and SAFIRE performed better than aNLM. For all organs, there was more than 85% agreement in organ-specific diagnosis among the three half-dose configurations and the full dose plus FBP configuration. Although a novel image-based denoising technique performed better than a commercial iterative reconstruction method in pediatric abdominopelvic CT examinations, it performed worse in pediatric chest CT examinations. A 50% dose reduction can be achieved while maintaining diagnostic quality.

Electron tomography (ET) offers a unique capacity to image biological structures in situ. However, the resolution of ET reconstructed tomograms is not comparable to that of the single-particle cryo-EM. If many copies of the object of interest are present in the tomograms, their structures can be reconstructed in the tomogram, picked, aligned and averaged to increase the signal-to-noise ratio and improve the resolution, which is known as the subtomogram averaging. To date, the resolution improvement of the subtomogram averaging is still limited because each reconstructed subtomogram is of low reconstruction quality due to the missing wedge issue. In this article, we propose a novel computational model, the constrained reconstruction model (CRM), to better recover the information from the multiple subtomograms and compensate for the missing wedge issue in each of them. CRM is supposed to produce a refined reconstruction in the final turn of subtomogram averaging after alignment, instead of directly taking the average. We first formulate the averaging method and our CRM as linear systems, and prove that the solution space of CRM is no larger, and in practice much smaller, than that of the averaging method. We then propose a sparse Kaczmarz algorithm to solve the formulated CRM, and further extend the solution to the simultaneous algebraic reconstruction technique (SART). Experimental results demonstrate that CRM can significantly alleviate the missing wedge issue and improve the final reconstruction quality. In addition, our model is robust to the number of images in each tilt series, the tilt range and the noise level. The codes of CRM-SIRT and CRM-SART are available at https://github.com/icthrm/CRM. Supplementary data are available at Bioinformatics online.

The ultra-high voltage electron microscope (UHVEM) H-3000 with the world highest acceleration voltage of 3 MV can observe remarkable three dimensional microstructures of microns-thick samples[1]. Acquiring a tilt series of electron tomography is laborious work and thus an automatic technique is highly desired. We proposed the Auto-Focus system using image Sharpness (AFS)[2,3] for UHVEM tomography tilt series acquisition. In the method, five images with different defocus values are firstly acquired and the image sharpness are calculated. The sharpness are then fitted to a quasi-Gaussian function to decide the best focus value[3]. Defocused images acquired by the slow scan CCD (SS-CCD) camera (Hitachi F486BK) are of high quality but one minute is taken for acquisition of five defocused images.In this study, we introduce a high-definition video camera (HD video camera; Hamamatsu Photonics K. K. C9721S) for fast acquisition of images[4]. It is an analog camera but the camera image is captured by a PC and the effective image resolution is 1280×1023 pixels. This resolution is lower than that of the SS-CCD camera of 4096×4096 pixels. However, the HD video camera captures one image for only 1/30 second. In exchange for the faster acquisition the S/N of images are low. To improve the S/N, 22 captured frames are integrated so that each image sharpness is enough to become lower fitting error. As countermeasure against low resolution, we selected a large defocus step, which is typically five times of the manual defocus step, to discriminate different defocused images.By using HD video camera for autofocus process, the time consumption for each autofocus procedure was reduced to about six seconds. It took one second for correction of an image position and the total correction time was seven seconds, which was shorter by one order than that using SS-CCD camera. When we used SS-CCD camera for final image capture, it took 30 seconds to record one tilt image. We can obtain a tilt series of 61 images within 30 minutes. Accuracy and repeatability were good enough to practical use (Figure1). We successfully reduced the total acquisition time of a tomography tilt series in half than before.jmicro;63/suppl_1/i25/DFU066F1F1DFU066F1Fig. 1.Objective lens current change with a tilt angle during acquisition of tomography series (Sample: a rat hepatocyte, thickness: 2 m, magnification: 4k, acc. voltage: 2 MV). Tilt angle range is ±60 degree with 2 degree step angle. Two series were acquired in the same area. Both data were almost same and the deviation was smaller than the minimum step by manual, so auto-focus worked well. We also developed a computer-aided three dimensional (3D) visualization and analysis software for electron tomography "HawkC" which can sectionalize the 3D data semi-automatically[5,6]. If this auto-acquisition system is used with IMOD reconstruction software[7] and HawkC software, we will be able to do on-line UHVEM tomography. The system would help pathology examination in the future.This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, under a Grant-in-Aid for Scientific Research (Grant No. 23560024, 23560786), and SENTAN, Japan Science and Technology Agency, Japan.

Dynamic CT myocardial perfusion imaging has the potential to provide both functional and anatomical information regarding coronary artery stenosis. However, radiation dose can be potentially high due to repeated scanning of the same region. The purpose of this study is to investigate the use of statistical iterative reconstruction to improve parametric maps of myocardial perfusion derived from a low tube current dynamic CT acquisition. Four pigs underwent high (500 mA) and low (25 mA) dose dynamic CT myocardial perfusion scans with and without coronary occlusion. To delineate the affected myocardial territory, an N-13 ammonia PET perfusion scan was performed for each animal in each occlusion state. Filtered backprojection (FBP) reconstruction was first applied to all CT data sets. Then, a statistical iterative reconstruction (SIR) method was applied to data sets acquired at low dose. Image voxel noise was matched between the low dose SIR and high dose FBP reconstructions. CT perfusion maps were compared among the low dose FBP, low dose SIR and high dose FBP reconstructions. Numerical simulations of a dynamic CT scan at high and low dose (20:1 ratio) were performed to quantitatively evaluate SIR and FBP performance in terms of flow map accuracy, precision, dose efficiency, and spatial resolution. Forin vivo studies, the 500 mA FBP maps gave -88.4%, -96.0%, -76.7%, and -65.8% flow change in the occluded anterior region compared to the open-coronary scans (four animals). The percent changes in the 25 mA SIR maps were in good agreement, measuring -94.7%, -81.6%, -84.0%, and -72.2%. The 25 mA FBP maps gave unreliable flow measurements due to streaks caused by photon starvation (percent changes of +137.4%, +71.0%, -11.8%, and -3.5%). Agreement between 25 mA SIR and 500 mA FBP global flow was -9.7%, 8.8%, -3.1%, and 26.4%. The average variability of flow measurements in a nonoccluded region was 16.3%, 24.1%, and 937.9% for the 500 mA FBP, 25 mA SIR, and 25 mA FBP, respectively. In numerical simulations, SIR mitigated streak artifacts in the low dose data and yielded flow maps with mean error <7% and standard deviation <9% of mean, for 30 × 30 pixel ROIs (12.9 × 12.9 mm(2)). In comparison, low dose FBP flow errors were -38% to +258%, and standard deviation was 6%-93%. Additionally, low dose SIR achieved 4.6 times improvement in flow map CNR(2) per unit input dose compared to low dose FBP. SIR reconstruction can reduce image noise and mitigate streaking artifacts caused by photon starvation in dynamic CT myocardial perfusion data sets acquired at low dose (low tube current), and improve perfusion map quality in comparison to FBP reconstruction at the same dose.