Using deformable models to register medical images can result in problems of initialization of deformable models and robustness and accuracy of matching of inter-subject anatomical variability. To tackle these problems, a novel model is proposed in this paper by compounding local invariant features and global deformable geometry. This model has four steps. First, a set of highly-repeatable and highly-robust local invariant features, called Key Features Model (KFM), are extracted by an effective matching strategy. Second, local features can be matched more accurately through the KFM for the purpose of initializing a global deformable model. Third, the positional relationship between the KFM and the global deformable model can be used to precisely pinpoint all landmarks after initialization. And fourth, the final pose of the global deformable model is determined by an iterative process with a lower time cost. Through the practical experiments, the paper finds three important conclusions. First, it proves that the KFM can detect the matching feature points well. Second, the precision of landmark locations adjusted by the modeled relationship between KFM and global deformable model is greatly improved. Third, regarding the fitting accuracy and efficiency, by observation from the practical experiments, it is found that the proposed method can improve  The majority of medical image processing tasks, including fMRI analysis However, there are still several critical issues to be resolved. Two recent surveys, The results of deformable model rely heavily on its initial position. If the initial position of the model is close to the region of interest, fitting results will be more accurate and the evolution time will be shorter. On the other hand, if the initial position is far away, it can lead to failure, due to the limited local searching algorithm of deformable models. Therefore, it may be worth investigating how to guarantee that the initial model is within the searching range. However, most attempts at improving deformable models have ignored this problem and left users to manually initialize it. Few research projects involve automatic initialization, and are limited to special applications  or uniquInter-subject anatomical variability may abolish one-to-one image correspondence between subjects. Thus global deformable geometrical model will not fit objects well, particularly when the same anatomical structures are absent in different subjects. Only a few methods have investigated this problem. In an attempt to address the problems mentioned above, we propose a novel compounding model by combining a deformable geometric model and local invariant features. Local invariant features can extract local structures robustly and consequently match them more accurately to images from different subjects. In this paper, we exploit the scale invariant feature transform (SIFT) Unlike other initialization methods mentioned previously, the proposed method can initialize global deformable geometrical models in more extensive ranges, due to its accuracy in matching local parts. Unlike Toews's model, the proposed model connects the local parts with explicit anatomical structures represented by PDM. Therefore the proposed method can find local parts among variable inter-subject anatomical tissues, as well as the meaningful anatomical structures.Extensively experiments have been carried out to evaluate the performance of the proposed method. We have collected data from a large number of cardiac MRI images, consisting of 500 slices from 16 subjects, 10 male and 6 female, aged from 45–64 years. Firstly, we demonstrate the matching accuracy of the proposed KFM with respect to its repeatability and robustness. Then the accuracy of initialization and fitting is evaluated. Finally, our fitting results show the The main conclusions of this paper is three-fold: 1) A KFM is built to match repeatable local parts among subjects with anatomical variance, even when no exact one-to-one correspondence exists. 2) An automatically initializing strategy is developed for the initialization of PDM. 3) An efficient strategy is developed to precisely localize landmarks of PDM.This study was approved by the Research Ethics Committee of Zhejiang University of Technology and Hangzhou Shaoyifu Hospital, China. All subjects used in this paper have been informed that their data may be published and provided written informed consent.The new model consists of a PDM, a KFM and their geometric relationship. The PDM is built in an ASM first and the gray-levels of pixels around the landmarks in the PDM are also modeled. The KFM is built by extracting SIFT features and choosing high-repeatable and high-robust ones. This section explains how to build PDMs and KFMs, and model their geometric relationship.A PDM consists of A new shape j-th landmark in the i-th image, j-th landmark form a gray-level profile, denoted by To find the accurate position of the new shape, the gray-level appearance should also be modeled. For each landmark, gray-level values are drawn from a line. This line passes through the landmark and is perpendicular to the boundary, which is formed by the landmark and its neighboring pixels. For Given total The first step is to align all The second step is to match the SIFT features to obtain highly-robust matching pairs. Usually, the appearance description of SIFT features is used to match SIFT features. SIFT features are local invariant features and their descriptions only represent their local image information. It may result in mismatched pairs, if there are similar local structures in two image but at different positions. After aligning SIFT features, an extra geometric constraint can be used to improve the matching correctness. Assuming two images in the training set are denoted as nth SIFT feature in the ith image being matched to the mth SIFT feature in the jth image.By using these conditions, SIFT features of each image are matched to all other images in the training set and a set of matched pairs can be obtained. We denote The third step is to choose highly-repeatable SIFT features by a matching propagation procedure. Given a SIFT feature, Supposing we finally obtain cth SIFT features in According to the three steps detailed above, the highly-robust and highly-repeatable SIFT features are obtained effectively. We can then use these SIFT features to build a KFM. A KFM consists of c-th SIFT feature in If a SIFT feature, cth SIFT feature in the kth SIFT feature set of KFM as lth landmark in PDM as The relationship between the KFM and the PDM is built through relative displacements between aligned positions of SIFT features in the KFM and aligned landmarks in the PDM. We denote the geometric position of the u-th SIFT feature Given a training image, the SIFT feature tth landmark, the position of this landmark in a test image can accordingly be determined by its displacement relative to the matched SIFT feature.Because in (8) positions of SIFT features in For each set of SIFT features in KFM, there are the relations denoted by (8) and (9), the position of the landmark in a test image is therefore determined by all SIFT features each of which is matched to one of the SIFT feature sets. It is worth noting that there may not be a matched SIFT feature for every In the proposed model, we only assess the appearance description and geometric position of local invariant features. However, it does not depend on special local invariant features. Therefore the proposed model can be easily integrated with other local invariant features, such as the maximally stable extremal regions (MSER) The proposed model can also be extended to the other deformable models with little difficulty. What the proposed model needs is a reference framework, just like the manually labeled landmarks in ASM. By using the reference framework, those extracted local invariant features can be normalized and the extra geometric constraint can be applied to improve the matching correctness. The AAM is similar to ASM and naturally has a reference framework since it also contains a manual labeling step. If using AAM instead of ASM in the proposed model, we can follow all the steps detailed previously. For ACM, an extra learning procedure is required. In this learning procedure, several reference points need to be labeled to normalize the local invariant features and the relationship between reference points and local invariant features will also be learned. The initial position of ACM can then be determined by the relationship between those matched local invariant features and interesting objects.For initializing the model in a test image, the SIFT features are extracted first and matched to the KFM. The first two matching conditions in (4) are used, which are similar to the strategies suggested in After obtaining an initialized model Assuming there are where ith new shape is computed by the following steps:After the precise position of each landmark in the PDM is computed, the same iterative process to ASM can be used to obtain the ultimate position. After obtaining a new shape jth landmark, in For one landmark, e.g. the Then we compute the difference between the mean profile Repeating the pervious two steps and obtaining a new set of points, denoted by Next we transform These iterative steps usually are time-consumed and need a hundred iterations to converge if the initial position of PDM is not ideal. By applying the proposed method, however, the number of iterations will significantly decrease because each landmark was located at its precise position. Thus the time cost will be sharply reduced.http://www.cs.ubc.ca/~lowe/keypoints/.We evaluate the proposed method in a set of cardiac MRI images. A large number of cardiac MRI images were collected for the experiments. The data consisted of 200 slices from 16 subjects, 10 male and 6 female, aged from 45–64 years. Five groups of experiments were carried out based on five randomly selected image sets. In each group, we randomly select 10 subjects and approximately 120 slices. Around 60 slices from 5 subjects were used for model training, and the remainder for testing. Examples of the experimental images are shown in As the proposed model is based on the highly-repeatable and highly-robust key points in the KFM, the repetition and robustness of the KFM are investigated first. In five groups of experiments, the numbers of key points in each KFM are 28, 26, 31, 20, and 22 respectively. To evaluate the repeatability of the KFM, the SIFT features were extracted from all images tested, and these features matched to the corresponding KFM. The number of times that correctly matching key points in KFM with test images were accumulated, allowing calculation of the repeated ratio for these key points. The minimum repetition ratio was 0.53 and the maximum repetition ratio was 0.96. The repetition ratios of 127 key points in all five KFMs are shown in Additionally, we calculated the maximum, minimum and average numbers of matching SIFT features in a new image. For further evaluating the matching correctness of KFM, we compute the average ratio of correct matching, which is defined as the ratio of correct matching points to all matching points for all test images. In all five KFMs used in the experiments, the maximum and minimum average ratios of correct matching are 1 and 0.91, respectively. We illustrate the average ratios in From Toews's model also matches features extracted from the new image As mentioned in the sub-section of Key Features Model, the picking ratio is an important parameter that balances the repetition and amount of key points in the KFM. It must be tested carefully and only the most proper ratio can be adopted. We evaluate several different picking ratios, such as 0.5, 0.6, 0.7 0.75, 0.8, 0.85 and 0.9. The number of key points and the repeated ratio under different picking ratios were computed respectively. All experiments in this section are based on the image dataset from the first experimental group.In From lth landmark point in the manually labeled shape of the ith test image manually labeled, lth landmark point in the resulting shape of initialization for the As the first step of the fitting process, the initialization is very important. The accuracy of initialization directly affects the fitting results. In the standard ASM, there is no strategy to accurately and automatically initialize the PDM. In our experiments, we achieve enough matched key points with a high enough accuracy to initialize the PDM by using the KFM. For evaluating the accuracy of initialization, we employ the average error which is defined in The ratio of correct initialization is defined as the ration between correct initialization of the PDM and the number of all test images. The correct initialization of the PDM is determined manually. Actually, only these initialized PDMs are treated as correct initialization, if the number of landmarks, whose distance to the corresponding manually labeled landmarks is less than five pixels, is more than half of landmarks in the PDM. During the process of pinpointing landmarks, the evaluation of precision is dependent on the distance between the position of manually labeling landmarks and pinpointing results by the proposed method. If the distance is less than three pixels, we believe that a landmark is precisely pinpointed. In all five experimental groups, the average number of precisely locating landmarks and the average precision are shown in We now evaluate the accuracy of the fitting results of our model by comparison to an improved ASM proposed in Firstly, as This paper proposed a method of a deformable model utilizing local invariant features for accurate and rapid registration of cardiac images. The highly-repeatable and highly-robust local invariant features are chosen from a training set to develop a KFM. The the relationship between the KFM and the PDM is obtained statistically. During the fitting process, the PDM is transformed to its initial position according to the transformed parameters, by aligning the accurately matched SIFT features between the KFM and new images. The precise position of the PDM is further achieved by modifying the landmark coordinates in the PDM according to the relationship between the KFM and the PDM. Finally, the ultimate location of the PDM is obtained by an iterative process with less subsequent reduction in computational load.Experiments demonstrate that this model outperforms traditional SIFT, in the sense of feature matching accuracy and robustness in cardiac MR images. Furthermore, it also surpasses other ASM regarding fitting accuracy and speed. Our experiments demonstrate that the proposed model improves the fitting accuracy by approximately 6–8% with only half of the computational cost of a state-of-the-art ASM.


Floating Skeleton concept suggests a biomechanical rationale for this newly observed phenomenon: that there exists a subperiosteal hydrostatic connection of synovial joints, and that this “net” distributes excess pressures among joints through the periosteal sheath to sustain the integrity of the joint contacting surfaces over a lifetime.Hydrostatic pressures can be transmitted between synovial capsules. In each of ten rabbits, we simultaneously measured pressure in two joints, one of which was passively ranged, and the other of which was kept stationary. The intra-articular pressure inside the stationary joint changed every time its companion joint was ranged. But the pressure in the stationary joint did not change when the periosteum was transected above the ranged joint. This phenomenon was observed in all four animals that served as their own controls. The study suggests that the intra-articular pressure was transmitted through the space between the periosteum and the bone surface. Alternative explanations, like measurements of venous blood pressure, did not show correlation with hydrostatic pressure changes in the joints. The  It has been documented in the past that if a joint is stationary, the hydrostatic pressure in its synovial capsule remains constant, and if the joint is passively bent or ranged, the pressure changes12467The purpose of the current study was to determine if the hydrostatic pressure variations in one joint are transmitted along the skeletal system to other joints, including distant joints. Simultaneous measurements in ten rabbits were made of intra-articular pressure in pairs of joints, one of which was put through a range of motion (passive flexion-extension) while the other remained stationary (motionless).In each animal, we paired joints both ipsilaterally and contralaterally, in the fore and in the hindlimbs. One of the joints was passively put through a cyclic flexion-extension while the other was immobilized and remained stationary . The exp12The new result, which to our knowledge has never been reported in the literature, is that pressure changed in the paired stationary joints in response to the ranging of the companion stationary joint, even when the pairs were contralateral.One explanation for the pressure change in the stationary joint is the possible mechanical action of the muscles and surrounding tissues caused by the cyclic bending in the ranged joint. This mechanical action may affect the shape of the synovial capsule of the stationary joint, and thus explain the change in intra-articular pressure. This conclusion may be correct, however, only for those joint pairs which are close to each other, like the ipsilateral Ankle-Knee, or Knee-Hip pair. This hypothesis cannot explain, however, pressure changes in a stationary distant joint, like the contralateral knee.Another explanation could be derived from the known phenomenon of correlation between synovial fluid pressure and the pressure in the blood capillary in the synovium3The third explanation for the phenomenon observed, and the one we find most compelling, is that the intra-articular pressure was transmitted hydrostatically through the subperiosteal space. To verify, pressures in the stationary knee were measured while the contralateral knee was ranged, both before and after the periosteum was transected 3 cm beyond the groove of Ranvier proximal to the passively ranged knee joint. Before the transection, the passive knee's pressure changed for all four animals taken for that experiment; after the transection, it changed for none of them . This ouIn future studies, the intra-articular pressure network should be more richly mapped. Simultaneous pressure measurements should be made in multiple capsules and along the periosteal sheath for a detailed mapping of pressure transmission from one synovial capsule to another. This network may provide new insight toward understanding the mechanism of intimate contact between the bone heads in a joint. In particular, it may help explain how joint heads safely withstand the surprisingly high reported values of contact pressures, which are close to the limits of cartilage integrityRaw data on pressures measured in the paired joints are presented in Pressure conductivity between joints was explored by measuring pressure changes in stationary joints while the joints they were paired with were cyclically bent , 2. The Fourteen (14), fifteen (15), fifteen (15), and twenty-two (22), observations were made for the Ankle-Knee, Ankle-Hip, Knee-Elbow and RKnee-LKnee pairings, respectively. In As a consequence of the exploratory analysis, it was necessary to perform a controlled confirmatory analysis to assess whether the changes in pressure could be explained by natural variation. First, a chi-square test (with three degrees of freedom) revealed that those four proportions did not differ statistically significantly from each other (p = 0.86), so one choice of pairings was not better than another for the confirmatory analysis.We opted to perform the control study on the contralateral RKnee-LKnee pairing, which circumvented the contribution of the muscles on the shape of the stationary synovial capsule. That contribution could not be ignored in the Ankle-Knee and Ankle-Hip pairs.In the control study, measurements were taken before and after the periosteum was transected distally to the ranged knee joint using the intramuscular approach. For this matched pairs study, the right knee and left knee (RKnee - LKnee) were paired. Along with the intra-articular pressure, we also measured the venous pressure , 4. The It was found that transecting the periosteum did not affect the pressure variations in the ranged joints . HoweverWe performed McNemar's test on the RKnee-LKnee observations. McNemar's test is designed for matched pairs, and formally measures whether row and column marginal frequencies are equalthe lowest possible p-value with a sample size of 4. With a sample size of 4, there was no power to detect a difference — that we found the lowest possible p-value for this design, however, implies the need to consider a similar study with a larger sample size.In all four RKnee-LKnee observations before transection, there were pressure changes in the stationary joint. In all four RKnee-LKnee observations after transection, there were no pressure changes in the stationary joint. The p-value for the test of equal effects was 0.13, Changes in venous pressure during trials were statistically insignificant and did not correlate with pressure changes in the stationary joints , 4.The fact that pressure in the stationary joints changed in response to the pressure variations in the driving joints, but never changed after the periosteum above the driving joint was transected, suggests that the transmission occurred through the muscle-skeletal system, specifically in the space between the periosteum and the bone surface. The potential role of the blood system in the detected pressure transmission was not supported by our experiments.The study revealed a phenomenon of variations of the intra-articular pressure in stationary joints in response to pressure changes in passively ranged jointsOne possible explanation of the phenomenon is related to the known link between intra-articular pressure and blood pressure in the synovium within single capsules. There are data on the influence of changes in blood pressure in the synovial membrane on the pressure inside that capsule2Could the vascular system be involved in transmitting pressures between multiple synovial capsules? Several steps would be necessary. First, the pressure changes in one capsule's synovial fluid would be transmitted via the blood plasma to the capillaries located in the capsule's membrane. This first step is quite possible based on the evidence reported above. The next step is difficult to account for, where the pressure gradient is transmitted via the vascular system to distant joints, for example from capsules of the left knee to the right knee. This passage from one capillary bed to the contralateral capillary bed would require passage through the entire circulatory system, which would disperse this gradient. By design, when pressure in the vessels rises or falls, their elastic walls stretch or recoil, changing the vessels' volume. This mechanism is responsible for the dissipation of the pulsation of arterial pressure in the capillaries, and further in the veins, and for the reduction of venous pressure compared with arterial pressure. Thus, even if a vascular contribution exists, it ought to be less significant compared to other routes.We still experimentally examined whether pressure might be transmitted through the vascular system. Venous pressure didn't show any correlation with the intra-articular pressures in either the stationary or the ranged joints. This discrepancy was likely because the mild correlation effect previous reported was for unhealthy rheumatoid joints and for more extreme conditions compared to our study, and, unlike in our study, certain agents were effused to the capsule3Another proposed explanation for the phenomenon observed is the mechanical action from the muscles which impact the position and shape of both joints. While a possible explanation for the closely paired joints like in the Ankle-Knee and Ankle-Hip pairs, the complication of joints co-affected by muscle action does not arise for the more distant pairs of joints, like contralateral knees. It was on these distant joints that we ultimately conducted our control experiments.In our control trials, the periosteum was transected 3 cm beyond the groove of Ranvier of the ranged knee (so above where the majority of capillaries are located)15The experimental results can be explained by hydrostatic conductivity between joints. Evidence comes in the form of pressure changes (Δp) in the stationary joints after distant joints are cyclically bent –4. AccorWe opted to directly measure pressure in joints instead of relying on radiology, which would require the dispersion of radioactive particles to the areas of interest. Also, radioactive particles are rapidly absorbed by and travel within the blood system.2O (1.4 mm Hg) due to acute flexion of the ankle joint in a rabbit. Tarasevicius, et al. reported the mean hydrostatic intracapsular pressure of 2.2 mm Hg in a human hip in 45° of flexionIn our analysis, a pressure transmission was considered to have occurred when a change in the pressure in the stationary joint was no less than ±2 Hg. That amount is in the range reported by other researchers who investigated variations in intra-articular pressure under different conditions. LevickThe ranging of a joint in our experiment, i.e. its passive flexion-extension, was performed manually, without instrumentation standardizing the angulation and bending moments exerted by the operator. As a result, in three trials we did not observe any pressure change in the ranged joint, attributable to an insufficient amplitude of bending. Data on these trials were excluded from the analysis. An occasional absence of pressure changes in a joint in response to its bending was explained by Wingstrand and colleaguesOur technique of measuring hydrostatic pressure with cannulae inserted into the synovial capsules was similar to methods used by other authors149A logical question arises about the morphological possibility for hydrostatic conductance between the capsules of distant joints. The periosteum lines the outer surface of all bones except the sesamoid bones and the intra-articular ends of bone2018At present very little is known about the response of the periosteum to the mechanical environment or to mechanical stimulation. However, with its modulus of elasticity in various parts in the range of 91.7+/−30.5 MPa and 63.0+/−25.4 MPa924A possible structure for providing and facilitating the hydrostatic connectivity between the synovial joints through the periosteum could be a system of gap junctions. A gap junction consists of intercellular channels providing aqueous continuity between adjacent cells ranging in width from 20 to 40 nm20As there is not yet sufficient knowledge about the morphology of the subperiosteal hydrostatic system we are discussing here, the authors commissioned a professional illustrator to create an artistic representation of this system in humans . A subpeWhile the rabbit animal model used in this study is widely accepted for investigating the human skeleton, the authors realize that primate studies might be more convincing. The latter animal model would be important to better understand the mechanisms that protect joints from overloading and for developing more effective prevention, treatment and rehabilitation modalities.Floating Skeleton concept introduced in 1993 by one of the authorsThe demonstrated transmission of pressure traces its origins to the The study presents the first experimental verification of the hypothesis of hydrostatic connectivity of synovial capsules via the periosteum. A new role is therefore suggested for the periosteum, in addition to the previously attributed roles of osteogenesis2331The static transmission of pressures along the skeleton could also suggest that the actual contact pressures between the cartilages in a joint are significantly lower than has been estimated with different methodologies. One such methodology is a computerized gait analysis representing the skeleton as a poly-linker with pin joints between solid linksAnother approach that produces suspicious results involves placing the load or pressure sensors in the contact zone of cadaver specimens or in artificial joint heads in subjects with joint replacement interventions. The pressures in gait on the heads of the instrumented artificial hip joints were found to be in the 5- to 6-MPa range and 18 MPa on average when rising from a chairAn indication of the role of the walls of an intact synovial capsule in redistributing pressure on the joint cartilages was provided in a recent cadaver study by Jaumard and colleaguesAll experiments were performed in accordance with relevant guidelines and regulations. The protocol for the study was approved by the IACUC of the Pine Acre Rabbitry Farm, Norton, MA, where all experiments were conducted. Ten male New Zealand rabbits weighing 3.5–4 kg were sedated and anesthetized with nitro gas and narcotic intra-venous.The study was designed to answer two questions: 1) whether the pressure in a stationary joint changes following pressure changes in the articulated joint; 2) if this change is recorded, whether the pressure is transmitted subperiosteally.To answer the first question we conducted a series of simultaneous pressure measurements in passively ranged and stationary joints. The joints were paired ipsilaterarly: Ankle-Knee, Ankle-Hip, Knee-Elbow, and contralaterally: Right Knee-Left Knee . The driThe timeline for the pressure changes and the venous blood pressure was recorded. A chart of time delay in pressure change response after the driving joint was first ranged is shown in As shown in Vitals including temperature, blood pressure, heart rate, and oxygen saturation were monitored throughout the procedure. The setup of the experiment for simultaneous measurements of pressure in a pair of joints is further illustrated in vastus lateralis are minimal in the distal part of the femur. We incised the muscle fascia, investing the vastus lateralis just anterior to the lateral intermuscular septum and elevated the muscle fibers off the septum, working from distal to proximal. This was most easily accomplished by use of a large elevator. We completed an atraumatic elevation of the vastus lateralis from the lateral aspect of the distal femur. We then placed retractors to give us visualization of the femoral shaft. We only needed a small window so we could make a circumferential incision in the periosteum. We then closed the fascia of the vastus lateralis with a running absorbable suture (vicyrl) and then closed the iliotibial band with a running absorbable suture. Skin was closed with nylon sutures. This is the same approach used in humans for femur fracture plating. However, since we were not plating the rabbit's femur but instead making a small circumferential incision in the periosteum, we were able to minimize the surgical dissection and trauma.To answer the second question of the study: if the pressure in a stationary joint changes following pressure changes in the articulated joint, whether that pressure is transmitted subperiosteally, we conceived a control experiment with four animals, measuring pressures on contralateral knees in which the periosteum was cut 3 cm above the ranged knee's groove of Ranvier . We madeMeasurements of the venous blood pressure were conducted to answer the question about the possible influence of the circulatory system of pressure transmission between the joint capsules. The rabbit's ear was chosen as the location for venous pressure measurement due to the large venous plexus present in the ears. Once the area was sterilely prepared and the vein identified, a catheter was introduced into the vein. A transducer was then attached to the catheter via intravenous tubing. Sterile saline was used to flush the line and check that there was a good flow via the tubing into the vein. The transducer was connected to the monitor device, the second DRE Waveline ProVet Multi-Function Patient Monitor, DRE Veterinary, Louisville, KY, as shown in Since the sensitivity of the apparatus, according to the manufacturer , was ±1 mmHg, we conservatively counted any reading under ±2 mmHg as 0, or as no change.The nature of our data informed the statistical methodology employed to analyze it. In our dataset, we have, for each animal and joint pair under consideration, a measurement of the difference in intra-articular pressure inside the passive joint (difference = pressure inside passive joint after other joint was articulated minus pressure inside passive joint before other joint was articulated). Some differences were negative; others were positive; some were equal to zero.any change in pressure – whether positive or negative – we considered as evidence of pressure transmission. A simple mean of pressure change would be an inadequate measure, since changes of, say,  are all interesting and are each indicators of synovial hydrostatic connectivity, but the mean of these 4 potential observations is 0.According to the presently accepted theory, the intra-articular pressure in the passive joint should remain unchanged (the before-after difference should be zero) when a distant joint is articulated, since their sacs of synovial fluid are isolated from each other. Therefore, With sample size as low as we had, we did not look at the absolute value of the difference in intra-articular pressure, since its distribution would have been non-symmetric, and symmetric and reasonably normal data are required for standard parametric t-tests when sample sizes are small.We therefore opted for a non-parametric approach to answer the chief question of our study: was there a change in pressure, or wasn't there? Each animal acted as its own control, and so that pressure changes in the same passive joint were measured before and after the periosteum was transected. If there were no mechanism of pressure transmission in the musculoskeletal system, then the presence or absence of pressure change should be unaffected by the transection: if pressure changes before transection, it does change after; and if it does not change before transection, then it should not change after.The pair of joints for the control study was to be chosen after exploratory analysis. If one pair of joints tended to produce a higher proportion of pressure changes than the other pairs, then we would study that pair in the confirmatory analysis, since a finding of no pressure change post-transection would be more surprising. If the joint pairs did not differ significantly in proportion of passive joint pressure changes, then we would just choose one. We opted for the contralateral Right Knee-Left Knee pairing, to overcome the issue of articular deformation in the passive joint caused by muscle contraction.To measure the agreement in pressure change before and after transection, we employed McNemar's test for paired dataExperiments with intact periosteumExperiments with cut periosteum


Regional heavy rainfall is usually caused by the influence of extreme weather conditions. Instant heavy rainfall often results in the flooding of rivers and the neighboring low-lying areas, which is responsible for a large number of casualties and considerable property loss. The existing precipitation forecast systems mostly focus on the analysis and forecast of large-scale areas but do not provide precise instant automatic monitoring and alert feedback for individual river areas and sections. Therefore, in this paper, we propose an easy method to automatically monitor the flood object of a specific area, based on the currently widely used remote cyber surveillance systems and image processing methods, in order to obtain instant flooding and waterlogging event feedback. The intrusion detection mode of these surveillance systems is used in this study, wherein a flood is considered a possible invasion object. Through the detection and verification of flood objects, automatic flood risk-level monitoring of specific individual river segments, as well as the automatic urban inundation detection, has become possible. The proposed method can better meet the practical needs of disaster prevention than the method of large-area forecasting. It also has several other advantages, such as flexibility in location selection, no requirement of a standard water-level ruler, and a relatively large field of view, when compared with the traditional water-level measurements using video screens. The results can offer prompt reference for appropriate disaster warning actions in small areas, making them more accurate and effective. When a large amount of water cannot be drained in time within the rainfall area, we face river overflow or urban inundation, which frequently causes a large number of casualties and a considerable property loss. Therefore, effective near real-time hydrological information is extremely important for flood warning and advance resident evacuation. Recently, there have been several studies on both forecasting- and monitoring-based flooding warning. At present, the major sources of the forecast estimation are large-scale remote sensing methods, including meteorology satellites and radar-based quantitative precipitation estimation. The forecast estimation utilizing multiple forecast models, the ensemble forecast technique can be used for performing simulations with integrated remote and on-site observation data; the analysis results can be further used for forecasting future precipitation or flooding information –5. HowevToday, all the developed countries in the world are using a variety of weather forecasting systems to assist disaster prevention, relief, and evacuation, in order to drastically reduce the number of casualties and the amount of economic loss caused by disastrous weather conditions ,6–9. Howin-situ water-level measurements, which not only provide important observation data for weather forecast systems for further analysis and prediction, but can also be used for providing instant flood warnings. The instant water-level monitoring techniques for precipitation mainly focus on the measurement of the relative height of the water surface. In general, four types of measuring sensors are used for quantitatively determining the water level: Pressure sensors, bubble gauges, float gauges, and non-contact radar gauges. These sensors are used for performing on-site measurements of the water level in rivers, drainage systems, sewers, flood storage ponds, and reservoirs  ×(%)Risk =[(c)If Risk > 80%, warning.where jA denotes the priori preset warning position point, which is set by technicians from the responsible unit according to specific views in different locations; j = 1:k represents the number of warning points, with the maximum value of k suggested to be 5 to 10. waterR refers to the identified overflow region in the image calculated by the proposed algorithm. The overflow region is shown as the white area in jA included in waterR is the current flood risk level. In other words, Risk is the percentage of alarm points overlapping with the water region. When the risk level is above 80%, the first warning should be sent. These warning can be superimposed on the Taiwan topographic map of the hydrological monitoring network. As shown in Flood risk detection and warning are included in the image-processing module to detect the risk level of the flood region and determine whether a warning should be generated on the basis of the risk level. Assuming that the flood is an invasion object and the entire field of view is the our surveillance view, the overlap proportion of the detected overflow region and the preset warning points are used as the standards of risk estimation. For example, if there are five preset warning points and four of them are invaded, or included in the overflow region, then the risk is 80%. 2.4.At this point, we have implemented the operational tools within a cyberinfrastructure platform, the image-based flood alarm (IFA) system, to monitoring the near real-time flooding level on the video streaming of remote cameras. What remains is the influence of atmospheric conditions that affect the image processing results. Severe atmospheric conditions exert complex visual effects for outdoor imaging, such as fog and raindrop stains. For the fog, the rainstorm involves suspended particles, mist, raindrops, raindrop streaks, and heavy rain spray; they add a non-uniform fuzzy mask between the scene and the camera render images extremely unclear. On the other hand, raindrop stains tend to adhere to the lens of cameras that refracts and reflects the light to generating shape and intensity changes in recorded images. These conditions considerably weaken the image quality of cameras and the outcomes of subsequent image processing. In proposed system, GrowCut exhibited superior resistance to rain stains, but yielded segmentation failure in the period of dense fog and the nighttime.3.The proposed flood detection system was tested using a real outdoor surveillance video case. The proposed video processing module and flood risk detection method were adopted in this test case to identify the flooding area and analyze the flood risk. The remote surveillance video used in this experiment was taken from 12:11 to 5:50 p.m. on 19 September 2010, when Typhoon Fanapi passed by. It was the peak rainfall period during Typhoon Fanapi, so the video recorded the sudden flood overflow caused by the heavy rainfall within a short time, at the Chongde Bridge section of the Erhjen River basin in the Tianliao district of Kaohsiung. The outdoor surveillance video system transferred the on-site real-time video back to the host computer via the Internet, and provided online monitoring, video archiving, and automatic video analysis for the water resource departments. On the host computer, screenshots were taken at 1-min intervals from all the surveillance video streams returned from the on-site cameras for a further analysis. The 1-min interval was set because the flood area usually does not change dramatically within 1 min and thus, the detection can still be considered a near real-time flood overflow detection with the 1-min delay. Moreover, the computational load and storage costs could be significantly reduced in this way. This real-life case covered a time period of about 6 h. A total of 350 images were therefore extracted for the experiment, and each of these JPEG images has a resolution of 352 × 288 pixels. Some of the tested images are shown in The proposed image-processing module can continuously perform segmentation for the input flood video stream by using the foreground and background seed points, and keep complete records of the flood region variations over time. The detected results for the flood objects will subsequently be used for the flood risk analysis. It has been experimentally proven that there is no stringent requirement on an accurate selection of seed points, which is in good agreement with the previous theoretical assumption. For example, the foreground seed points only need to be placed in the possible flooding area instead of the background. Therefore, the same flood detection result can be achieved without a 100% precise setting of seed point locations. The same is true for the background seed points. However, note that the background seed points have to be placed in locations that are unlikely to be reached by the flood when the water level increases. The algorithm used in this study only treats the seed points as the starting point of the initial image labeling and the final segmentation result after the iterative CA competition process is not restricted by the location of initial seed point but depends on the overall properties of the foreground and the background instead. However, the outward growth of the foreground may be slightly weakened if the background seed points are too close to the foreground seed points in practical cases, which will result in a withdrawal of the foreground boundary near the background seed points. Therefore, the use of prior tests or experience to set up the seed points will be helpful in obtaining a better and more complete segmentation of the flood area.All the real-life images were processed by the image-processing module to detect the flood object area. The flood detection results of this practical case are shown in With the automated image processing method, a large number of remote surveillance videos at various locations can be simultaneously monitored for a flood warning service. This can be combined with river monitoring images and city traffic surveillance images to perform large-scale near real-time flood detection and obtain information about the overflow area. Warnings are subsequently sent to the relevant staff on duty and online disaster prevention monitoring systems if the risk levels of the rivers or the other sections are higher than the threshold values. An automated, quick, and easy flood warning service can be provided to replace the manned monitoring. 4.The main purpose of this study was to improve the traditional surveillance image-based water level analysis and its relevant techniques for the detection of floods, overflow, and inundation. Traditional image-based techniques have been restricted by a number of disadvantages, such as the location choice of the ruler setup, the lack of a large field of view covering sufficient geographic information, the precise calibration between the camera and the ruler, the maintenance requirement for maintaining the fixed calibration status, and the scale conversion from the image size to the real-world size. This study proposes the use of the object intrusion principle to monitor a flood or inundation event and analyze the corresponding risk level. It has been proven by using a practical test case that the proposed methodology can correctly detect the changes in flood overflow and estimate the near real-time flood risk levels on the basis of the degree of flood intrusion.Traditionally, when a remote surveillance video is used for detecting the water level, the primary principle is to obtain the overlapped position of the water surface and the height on the ruler ,26 or thin-situ visible geographic information for visual verification. Eventually all the measured data can be easily confirmed by visual verification. All these advantages make up for the shortcoming of obtaining only the water level height information from previous image-based methods. Therefore, this proposed method has a relatively high robustness in outdoor flood detection and warning applications.As expected, the practical test case revealed that the outdoor image-based application is influenced by weather conditions and atmospheric visibilities. In particular, during heavy rainfall or night time, camera lenses that rely only on visible light are more easily affected by atmospheric visibility, which is consistent with our previous study . In addiThis study proves that the video surveillance theory can be used for providing an unattended flood detection and warning service. In addition, this automated detection system can perform a near real-time analysis on a large number of remote surveillance videos at the same time and provide an automated, quick, and convenient flood warning service. Similarly, it can be widely used in urban inundation detection. Combined with the currently existing traffic and security surveillance videos, it can be employed to automatically monitor the overflow of a city's inner river or the inundation of streets, which will help citizens stay away from the inundation regions and reduce the hazards brought about by urban inundation.


Philosophical Transactions B in 1971, Crowther described new computational procedures applied to the first three-dimensional reconstruction of an icosahedral virus from images of virus particles preserved in negative stain. The method for determining the relative orientation of randomly oriented particles and combining their images for reconstruction exploited the high symmetry of the virus particle. Computational methods for image analysis have since been extended to include biological assemblies without symmetry. Further experimental advances, combined with image analysis, have led to the method of cryomicroscopy, which is now used by structural biologists to study the structure and dynamics of biological machines and assemblies in atomic detail. This commentary was written to celebrate the 350th anniversary of the journal Philosophical Transactions of the Royal Society.Elucidation of the structure of biological macromolecules and larger assemblies has been essential to understanding the roles they play in living processes. Methods for three-dimensional structure determination of biological assemblies from images recorded in the electron microscope were therefore a key development. In his paper published in  Since then, the microscope has been one of the most powerful tools for extending the reach of the senses in both the life sciences and physical sciences. On a much smaller scale, the visualization of viruses, the causative agents of disease in humans, animals and plants, awaited the twentieth century development of the electron microscope, which can illuminate specimens with radiation of wavelength more than 100 000 times smaller than that of visible light. Viruses are assemblies that package their genetic code as nucleic acid, either DNA or RNA, for transfer from a host cell to their next target of infection. The electron microscope showed that while some types of viruses were variable in structure, others were remarkably uniform, either as rod-shaped or spherical particles. Structural studies of viruses and viral proteins by electron microscopy and X-ray crystallography have complemented biochemical and genetic approaches in understanding the life cycle of viruses, a subject important to both basic science and medicine.R. A. Crowther's ‘Procedures for three-dimensional reconstruction of spherical viruses by Fourier synthesis from electron micrographs’  describe2.Philosophical Transactions and in related publications with colleagues were performed at the MRC Laboratory of Molecular Biology (LMB) in Cambridge, where during the course of his career he was a graduate student, research group leader and Joint Head of the Structural Studies Division. The LMB has an illustrious history of achievement in macromolecular studies including X-ray crystallography, the first method to reveal the atomic structure of proteins, and many of the developments in electron microscopy discussed in this essay happened there. In 1972, around the time of the spherical virus work, Crowther described the fast rotation function in X-ray crystallography, a procedure for finding the orientation of protein models in crystals [The studies described by Tony Crowther in a). Purified TBSV particles are so regular that they form well-ordered three-dimensional crystals with cubic-shaped unit cells implying that each virus particle is identical in structure. In 1956, Crick & Watson [Nature, Don Caspar's X-ray photograph of a slice of the three-dimensional diffraction pattern of a TBSV crystal showed intensity spikes indicating an arrangement of fivefold, threefold and twofold symmetry axes consistent with the symmetry of the icosahedron [Tomato bushy stunt virus (TBSV) is a small, spherical virus that has featured as a prototypical macromolecular assembly in several landmark studies in structural biology, including Crowther's first work on virus reconstruction ,3,4. In figure 1During the 1960s Klug, Finch and co-workers studied the architecture of virus capsids by imaging viruses in the transmission electron microscope using negatively stained specimens . In the Their structural studies of viruses in stain also focused on a more detailed understanding of how the electron images were formed. By comparing images of virus particles that were sitting in stain on only one side versus those that were stained on both sides, they learned that images of virus particles uniformly embedded in stain were super-positions of the top and bottom of the capsid shells . This isb) shows strong intensities along the directions of the symmetry axes, consistent with Caspar's [Further insight into projection images in the electron microscope came from the analysis of rod-shaped viruses, such as tobacco mosaic virus (TMV), understood to be a helical organization of identical protein subunits that assemble around its RNA genome . Owing tfigure 1Caspar's  observatProtein crystallographers had learned that the diffraction patterns recorded while illuminating different orientations of a crystal were portions of a single three-dimensional lattice or diffraction pattern. The three-dimensional diffraction pattern is related to the structure of the protein in the crystal through the mathematics of the Fourier transform. There is a direct mathematical or computational procedure to determine the structure of a molecule from its three-dimensional Fourier transform called Fourier inversion.In the years immediately before Crowther's work on spherical viruses, a breakthrough occurred in the interpretation of projection images from the electron microscope through Fourier analysis, enabling the direct calculation of the three-dimensional structure or ‘reconstruction’ of the helical tail of T4 bacteriophage . The int3.et al. [Nature describing reconstructions of both TBSV and human wart virus. The paper presented at the Royal Society meeting was a description of new methods for reconstruction with TBSV as an application. Crowther acknowledged Linda Amos, with whom he co-authored several papers at this time, for writing computer programs for the new procedures. Two approaches were used for recording the views required for reconstruction. In the case of the human wart virus, two views of each particle were obtained by tilting the specimen holder through a known angle, a forerunner of the multiple tilts used to collect data for tomography of non-symmetric specimens [At the time of the Royal Society meeting, Crowther et al.  had submpecimens . In the The non-helical, spherical virus specimen presented a new problem. Images corresponding to different projections of the virus need to be combined in one reconstruction. This requires determining the orientation of different virus particles within the images and centring them in a single coordinate system. The specimen was a good candidate because the virus has distinguishable features in negative stain, and earlier evidence strongly indicated that it has icosahedral symmetry. A simple formula described the minimum number of views required to calculate a structure of a given diameter to a desired resolution. If an image could be assigned an orientation, then by application of the symmetry of the virus many more views of the virus could be calculated. Because the Fourier transform of an image represents a slice through the three-dimensional Fourier transform of the virus, then the three-dimensional Fourier transform can be filled up by a few images (five for the TBSV study), each with up to 59 symmetry-related views. The structure of the virus could then be calculated by an inverse Fourier transform as with crystals or helical specimens.Thus, the key issue in the analysis of single particles is to assign the orientations. Crowther observed that any two slices of the three-dimensional Fourier transform have a geometric intersection through a line where each transform would have the same values, referred to as a ‘common line’. In principle, the computer can search all the lines in two slices to find the common line, but this is very difficult for noisy, imperfect images. However, the symmetry of the virus can be exploited: one common line implies the existence of other common lines within a single image. A search for the number and location of many ‘self-common lines’ uniquely identifies the orientation of the icosahedral symmetry axes of the particle observed in the projection. The best assignment of a particle's orientation is one that gives the greatest numerical agreement between all common lines predicted for that orientation. Approximate values for the orientation and position of the particle in the image can be further improved by ‘refinement’. Several refined images can thus be consistently oriented in the same coordinate system.et al. [A number of significant computational issues needed to be solved to calculate the structure from separate projection images, including making sure that the data are sampled finely and regularly onto the final grid or volume on which the structure is calculated and presented. Some of the issues were discussed in an earlier work of Crowther et al. . The meta) showed how 90 morphological units, each explained as a dimer of the 180 capsid monomers in the virus, were arranged with respect to the icosahedral symmetry elements as well as local symmetry elements. The paper contained, for the first time, several recognizably modern demonstrations of the success or reliability of the reconstructions. Calculated projections of the reconstruction agreed with the experimental images (b). The paper gives a measure of the reconstruction quality (the agreement between matched common lines) as a function of resolution and showed a correlation to the expected resolution limit of negative stain (approx. 30 Å). This is sufficient to confirm the assumption of icosahedral symmetry and that the alignment methods are working. The modern expert will also recognize a number of other familiar features such as the enforcement of a consistent absolute hand and magnification as determined from the ‘cross common lines’ between particles.The map of the virus calculated in this way a showed l images b. The paIn short, the paper had accomplished three-dimensional image reconstruction from two-dimensional images of non-crystalline single particles. Spherical viruses are an ideal model system for developing methods, and though not the most general specimen, they are an important generalization from helical or crystalline specimens. Because of the limitations of the large grain size of negative stain and the small number of images, the structure of TBSV could only be determined to very low resolution. The paper was an important beginning to the single particle reconstruction field and a glimpse of the road ahead. While Fourier-based methods have been widely developed and applied, alternative iterative approaches to structure determination from projections in real space ,20 have c). The structure showed how the capsid protein uses two flexibly linked domains to adopt the different conformations required by quasi-equivalent packing. Radiation-sensitive virus crystals, many weak reflections and the difficulty of solving the crystallographic phase problem for a large molecular assembly posed huge technical challenges and led to important new methods in crystallography. Yet, the symmetry of the virus posed constraints on the density that were used to solve the structure. The properties of TBSV, its regularity and symmetry, were exploited in both electron and X-ray studies. Thus, TBSV and other spherical viruses helped to drive the development of ideas and methods in both fields [The atomic model for TBSV, the first high-resolution view of a crystalline virus, was determined at 2.9 Å by Stephen Harrison and co-workers using X-ray crystallography  figure c. The sth fields –28. In t4.Progress in the field of three-dimensional electron microscopy resulted from many developments in each of the three areas described at the 1970 Royal Society meeting. As anticipated in the conclusion of Crowther's paper, structure determination from individual particle images could be extended to particles with lower symmetry or even to proteins with no symmetry at all. For an asymmetric particle, many more views are required and the specific methods employed by Crowther for determining orientations could not be applied. Furthermore, images of particles in negative stain consist of desirable signal from the particle as well as noise that comes from the underlying structure of the carbon film support, particle distortion, stain variation and the photographic process. But by averaging similar views of a particle, the common signal of the particle in the images is increased compared with that of the noise, resulting in a higher signal-to-noise image. The ribosome, a large asymmetric macromolecular assembly of central biological importance as the engine of protein synthesis, was an excellent target for electron microscopy and drove the development of methods for studying asymmetric particles. The key developments included methods for statistical classification and alignment of similar views of the particles so that many high signal-to-noise averages of the particle can be obtained . In addiHalobacterium halobium. In 1975, Henderson & Unwin [High-resolution structure determination by electron microscopy was first demonstrated for bacteriorhodopsin (BR), the light-driven proton pump that forms crystalline patches in the purple membrane of  & Unwin  calculatThe work reported several major methodological advances. They studied unstained specimens in glucose, which effectively replaced the water in the sample but preserved native protein structure to high resolution. The resulting maps of the protein show density variations in the structure of the protein itself, including its interior, rather than the stain distribution. Such thin biological specimens are composed of light atoms and require small amounts of defocus during imaging to enhance their contrast, typically between a few hundred nanometres and up to a few micrometres. Furthermore, biological specimens without stain are extremely sensitive to irradiation by the electron beam, more so than the negative stain preparations discussed so far. The fading of the crystalline patterns of BR showed that images should be recorded with less than five electrons impinging on a square Ångström of the specimen to preserve high-resolution features. These studies therefore employed imaging methods that minimized the exposure of each crystalline specimen to a single ‘low dose’ image. For low dose images, statistical variation in electron counting is a major source of the noise. The diffraction patterns calculated from the images are the computed average of the scattering of all the molecules in the crystal. Analysis of the patterns is similar to averaging images of all the individual molecules in the crystal, thus overcoming statistical noise and giving a high-resolution image of the molecule.In subsequent years, Henderson and colleagues developed experimental and computational methods for analysing two-dimensional crystalline specimens to high resolution, ultimately obtaining a map for BR at 3.5 Å resolution, sufficient to build an atomic model . Data coThe breakthrough in sample preparation described by Jacques Dubochet and co-workers, following earlier attempts to prepare frozen-hydrated specimens , provideThe near atomic resolution information obtained from two-dimensional crystals and some helical specimens showed that high-resolution protein structure determination was possible with electrons, and the number of molecules in the crystalline lattice gave an indication of how many images of individual, unstained protein molecules would be required to reach atomic resolution. However, the molecules in crystals have the benefit of being aligned to each other, and their orientation is known from the easily measurable orientation of the lattice. In single particle analysis, each molecule's position and orientation must be determined in the image. The ability to find the orientation of a particle depends on whether it is large enough to scatter electrons strongly compared with the background noise, i.e. how visible it is. A theoretical calculation showed that for images limited only by the noise associated with low electron dose, 10 000 particle images should be sufficient to reach atomic resolution (3 Å) for a single particle of any size , though Nature by two groups [Instruments, sample preparation and image analysis were now poised for high-resolution studies of single particles. Symmetric viruses were the most likely to get there first because they are highly visible in amorphous ice due to their large size and are therefore easier to align. Aligning and averaging icosahedral particles includes 60 times more monomers or ‘asymmetric units’ than for an asymmetric particle. An important milestone was achieved by imaging a virus sub-particle, the icosahedral capsid assembly of hepatitis B virus, published in o groups ,43. In to groups . The calo groups  shown ino groups  presenteFor many years, single particle images have been recorded on photographic film, which captures high-resolution detail, and, for example, was the recording medium in the atomic resolution virus studies mentioned above. When electronic detectors (phosphor-based CCD cameras) became available, they were used widely because of their speed and convenience, even though film was a better detector. In the last couple of years, a new generation of direct electron detectors (DDD's or Direct Detector Devices) has been applied to imaging single particles, producing better images than film . In addiThe asymmetric 1.9 MDa 54S subunit of the ribosome, much smaller than the viruses described at high resolution, could now be solved close to 3 Å by electron microscopy and from fewer particles than previously imagined  as shown5.in silico because the alignment and averaging of images is done by the computer and no longer requires the growth of well-ordered crystals of the specimen. Microscopists have not yet reached atomic resolution for the lowest molecular weight limit or smaller number of particles predicted by theory [It would appear at the time of writing that many of the major obstacles to the atomic resolution imaging of non-crystalline biological specimens by cryoEM have now been surmounted, some of them in the last year or so. Electron microscopy is a technique with general applicability to a range of specimen types, including single particles, fibres and two-dimensional crystals. One hundred years after Bragg's Nobel Prize for the development of X-ray diffraction analysis, single particle cryoEM joins X-ray crystallography and NMR spectroscopy as a high-resolution structural method for biological macromolecules. Because of its similar goal to X-ray crystallography, the single particle approach has been described as crystallization y theory , but aveFuture developments of three-dimensional electron microscopy with impact in biology will include further generalization of the types of specimens that can be imaged at high resolution. Even with recent rapid advances, high-resolution single particle analysis is largely restricted to proteins that are biochemically purified and are assumed to be nearly identical in structure. In the future, electron microscopy will include many more investigations where these assumptions can be relaxed to include specimens with structural flexibility and heterogeneity. The function of many molecular machines is achieved through large structural transformations. The classical approach is to trap the molecules in a well-populated intermediate for imaging. But by breaking the molecular machines out of crystalline lattices, we can hope to image the structural transformations happening in solution. Examples include the membrane rotary ATPases, whose motion is linked to the synthesis or hydrolysis of ATP , and theOf course, when a specimen has conformational or compositional heterogeneity, it is more complex to tell a computer how to classify and average particle images. Any particle image can be assigned to one of several different structures and to different orientations. Each member of the ensemble of structures requires an independent structure determination and many more particle images are required for the whole multiple structure dataset. Statistical approaches to image processing such as maximum likelihood have contributed to the resolution revolution , but at in vivo [While symmetrical viruses can be solved by single particle methods, many viruses that are important human pathogens are variable in structure or pleomorphic, such as the lipid-enveloped influenza viruses. Each virus is different, and images of different viruses cannot be averaged together, and they are best studied by cryotomography. In tomography, all the unique projection images required to determine the structure by electron microscopy are recorded from a single specimen as a tilt series, and the resulting three-dimensional reconstruction is called a tomogram . There iin vivo .Transactions published a paper presented by Nigel Unwin at the 1970 Royal Society meeting that contained a description of a phase plate consisting of a thin spider web coated with metal suspended across the objective aperture of an electron microscope, and this produced phase contrast images of TMV [Where molecular averaging is not possible, there may still be opportunities for higher-resolution studies through improved instruments and improved image analysis. The defocus mechanism of contrast used in cryoEM at present is imperfect because the contrast is not uniform across all resolution ranges. Zernike's phase plate for the light microscope had been proposed by 1940 and has been used in light microscopy for many years. Phase plates for electron microscopy are currently an active area of research and could help record high-contrast, in-focus images. Interestingly, the s of TMV . Though s of TMV .Micrographia tell us what he saw through the eyepiece. The development of camera technology and computational methods in microscopy has allowed a more detailed and objective analysis of the specimen. In electron microscopy, image analysis revealed molecular structure by first exploiting the special features of crystalline and helical specimens and then finding ways to analyse less periodic specimens, as exemplified by Crowther's paper. Advances with prototypical specimens revealed how instrumentation, sample preparation, data collection and analysis could be improved to solve the structure of the most general, asymmetric specimens. Our ability to collect vast image datasets and analyse them rapidly by computer will no doubt extend our understanding of biological structure and function. Images are now just frames in a movie of the specimen's lifetime. Computational image analysis has become an essential lens through which we observe and attempt to understand the movies of the asymmetric and dynamic structures we call life.There has been a history of improvement in the instrumentation used to image biological macromolecules while the means to record the image and analyse its features has similarly undergone dramatic changes. Robert Hooke's spectacular drawings in


The properties of many biological materials often depend on the spatial distribution and concentration of the trace elements present in a matrix. Scientists have over the years tried various techniques including classical physical and chemical analyzing techniques each with relative level of accuracy. However, with the development of spatially sensitive submicron beams, the nuclear microprobe techniques using focused proton beams for the elemental analysis of biological materials have yielded significant success. In this paper, the basic principles of the commonly used microprobe techniques of STIM, RBS, and PIXE for trace elemental analysis are discussed. The details for sample preparation, the detection, and data collection and analysis are discussed. Finally, an application of the techniques to analysis of corn roots for elemental distribution and concentration is presented.  The study of biological processes in living organisms shows that many important functions depend on the presence of specific essential/trace elements. The essential trace elements can be defined in simplest form as that element which is required in small quantities for the maintenance of life. Its absence or excessive presence beyond the right amounts results in either death or malfunctioning of specific organ in the living organism.Out of all the naturally occurring elements, about 17 and 26 are known to be essential to plant life and animal life, respectively. For an element to be essential for plant growth, it must meet two main criteria in accordance with Liebig's law of minimum, a principle developed in agricultural sciences by Carl Sprengel (1828) and later popularized by Justus von Liebig, which states that growth of a plant is controlled not by the total amount of resources available but by the scarcest resource [The significance of the essential trace metals in biological systems is indisputable due to their positive roles when in specific concentration ranges and toxic roles in relatively high or low concentration levels. The essential trace metals have four main functions which include (i) stabilizers, (ii) elements of structure, (iii) essential elements for hormonal function, and (iv) cofactors in enzymes. Inadequacy or lack of trace elements will affect the structure alone or will affect structural function due to lack of stabilization, change of charge properties, and allosteric configuration . An exceμm or less), essentially all incident protons which are not backscattered will be transmitted. STIM images are generated by measuring either the transmitted ion energy or variations in the number of ions at each image pixel within the scanned area. Only one ion per pixel is required in principle to measure the energy loss, but, in practice, owing to energy straggling, several ions are required to generate low noise images. The best method of generating STIM image is using an event-by-event data acquisition system. The measured data set of the ion energy loss values at each pixel can then be manipulated in different ways to give the best image contrast. With average processing, the outlying events in the energy spectrum at each pixel, arising from slit scattering or other forms of noise, have a large effect on the average energy loss and so can introduce noise into the image. This can be avoided by discarding the outlying events. With median processing, the measured energy loss values in each pixel are ordered by increasing energy loss, and the central value is chosen. This defines the location of edges present within a pixel and effectively eliminates noise in the image. The density structure of the sample can thus be determined by measuring the energy loss of the transmitted beam particles [STIM is used to identify regions of interest in the sample. STIM is often used to investigate samples thin enough to transmit a 2-3 MeV proton beam. For thin organic samples  levels. PIXE is based on the ejection of an inner shell electron out of its orbit by an interaction with MeV ion. As the vacancy is filled by an outer shell electron, an element and shell-specific X-ray is emitted. The energies of the X-rays are characteristic of the elements in the sample and the intensities of the X-rays can be used to calculate the abundance or concentration of an element. It is the most commonly used microprobe technique and has been widely applied in trace element analysis in biomedical and geological fields .  Figure−7 seconds before being filled by an electron transition from an outer shell with a subsequent emission of either X-ray or an auger electron. The energy of the emitted X-rays is unique to the element, so the measured X-ray energy of the spectrum allows the elements present in the sample to be identified. With PIXE, the measured X-ray yield is nearly independent of the chemical state or bonding within the sample and the X-ray production cross sections are well known; therefore, trace element concentration of less than 1 ppm can be detected and quantified [When a typical MeV ion strikes a sample atom, the interaction generates a vacancy in the inner shells of the atom. MeV light ions have high cross section for ejecting K, L, or M shell electrons because their velocities approach the inner shell electron velocity. An inner vacancy exists for about 10antified .The X-ray signal from the trace elements of interest can be partially or totally masked by an unwanted background in the spectrum arising from the backing or host material. This background can be in form of a continuum due to bremsstrahlung or it may contain discrete peaks arising from interfering characteristic X-ray from the matrix material. The decrease in the continuum at the lowest energies is due to absorption of X-rays in the Be window of the X-ray detector. The background at low energies prevents easy and precise analysis of light elements in the spectrum. This is why PIXE is often used for measuring trace elements of mass above sodium.The ion beam intensity necessary for the quantitative analysis and normalization between sequential irradiations relies on accurate charge measurements. The current integration which is used to obtain charge can be determined by measuring the current on the conducting target or through the target on a faraday cup for thin targets. In case of insulating target, the current can be measured by evaporating or coating a thin layer of conducting carbon layer on the front surface of the target or by mixing a conducting powder with the target material in a predetermined quantity. The chamber is always biased appropriately to avoid the effect of secondary electrons on the charge measurements.μm polyethylene filter was interposed in front of the detector to stop the backscattered protons. The detector profile was set up in GeoPIXE and the corresponding detector efficiency measured as shown in The High Purity Germanium- (HPGe-) detector was acquired for PIXE analysis of biological samples. The HPGe-detector was manufactured by Canberra Electronics and it is an ultralow energy detector model, GUL0110, that came equipped with ITRP type preamplifier. For absolute quantitative analysis to be performed, the detector efficiency must be accurately known over a given energy range large enough to encompass all typical characteristic X-rays of interest in the sample . With thμm thick and quickly freeze-dried. The samples were then irradiated with a 2 MeV focused proton beam of resolution of 5 μm. Simultaneous RBS and PIXE were done and data collected. The RBS data was analyzed by SIMNRA [The seeds of corn were germinated in vitro in the dark for a period of six days in agarose gel. The root was excised 5 mm long and inserted in a plastic tube filled with freezing medium and then quickly cryofrozen in a container of isopentane (2-methylbutane) cooled with liquid nitrogen, which provides superior cryogenic condition without Leidenfrost phenomenon (boiling of liquid nitrogen). The root was then cryosectioned 60 y SIMNRA  to detery SIMNRA .Yk calculations include absorption and secondary fluorescence contributions. The result is a standard less quantitative method with minimum detection limits down to 1 ppm level. For the elemental maps and concentration analysis, a dynamic analysis (DA) matrix of the fitted PIXE spectrum was generated and used to generate the elemental maps. The spectrum peak areas ak are related to element concentration Ck by the following equation:Q is the integrated beam charge, Ω and εk are the detector solid angle and efficiency, Tk is the X-ray absorber attenuation, and Yk is the generic X-ray yield . Yk is assumed to be constant for element k across the entire image. The solution of the linear least-squares problem can be cast as a matrix equation that transforms directly from spectrum represented by a vector S to concentration vector C, which includes all detected elements in terms of the matrix Γ [ Agathosma betulina (Berg.) Pillans, colonized by Cryptococcus laurentii [The GeoPIXE program used for the analysis in this project has an inbuilt ability to extract the X-ray yields from the spectrum via peak fitting. The yield matrix Γ , 14:(2)Caurentii . The micThe average concentrations of the elements in the whole sections of the root were determined. The distribution of the elements in different regions of interest (ROI) of the root, whole root, the epidermis, the cortex, the endodermis, and the vascular tissue, was also determined and the 95% confidence interval of the average elemental concentrations calculated. The standard deviation of the mean for the elemental concentrations was also determined. The results were recorded as shown in  Sclerophyll betulina (Berg.) Pillans [The distribution and quantification of trace elements in different sections of the corn root were successfully determined. The preferred accumulation sites and the highest levels of P, S, K, and Fe were detected in the epidermis and endodermis sections of the root. We also noticed reduced concentration of these elements in the cortex and vascular tissue sections of the corn roots. The elemental concentration of the other detected elements including Cl, Ca, Ti, Cr, Mn, Ni, Cu, Zn, and As was generally low. This could be attributed to low statistics or low absorption of these elements by the corn roots. Their distributions were much similar to the more abundant groups of P, S, K, and Fe discussed earlier. Similar observations were made by Cloete et al. in their study of elemental distribution and mapping of roots of a Mediterranean-type Pillans , in whicNearly all the elements detected were primarily localized close to the symbiont exchange interface and within the epidermal and endodermal root tissues. This distribution pattern may be attributed to the presence of the apoplastic barrier present in the endodermis and the epidermis cell walls of root tissues, which tend to restrict the intertissue transport of certain elements . HoweverFor this biological sample of corn root, the nuclear microscopy was successfully used to map and quantitatively measure the elemental concentrations and distribution within the sample. This technique has proved to yield reliable results that can be extended to the analysis of other biological tissues. PIXE analysis consists of two parts as demonstrated. The first part is to identify the atomic species in the target from the energies of the characteristic peaks in the X-ray emission spectrum as shown in 15  on noninterfered low-background isotopes. This is achieved by ionizing the sample with inductively coupled plasma and then using a mass spectrometer to separate and quantify those ions. Compared to atomic absorption techniques, which is an electroanalytical procedure for the quantitative determination of chemical elements using the absorption of optical radiation (light) by free atoms in the gaseous state, ICP-MS has greater speed, precision, and sensitivity. However, compared with other types of mass spectrometry, such as thermal ionization mass spectrometry (TIMS) and Glow Discharge Mass Spectrometry (GD-MS), ICP-MS introduces a lot of interfering species: argon from the plasma, component gasses of air that leak through the cone orifices, and contamination from glassware and the cones. The nuclear microscopy techniques are capable of multielemental mapping and analysis of biological materials as demonstrated in this work. A large number of elements can be mapped and quantitatively analyzed simultaneously and with the help of a suitable software like GeoPIXE, the elemental distribution within the sample can be accurately determined. The technique is sensitive enough and has a minimum detection limit down to 1 ppm and even lower. This tool is also nondestructive making it uniquely suitable for biological analysis.The use of nuclear microscopy techniques shows a proven analytical tool that is competitive with other classical methods like inductively coupled plasma mass spectrometry (ICP-MS)  and atom


Acute viral infections pose many practical challenges for the accurate assessment of the impact of novel therapies on viral growth and decay. Using the example of influenza A, we illustrate how the measurement of infection-related quantities that determine the dynamics of viral load within the human host, can inform investigators on the course and severity of infection and the efficacy of a novel treatment. We estimated the values of key infection-related quantities that determine the course of natural infection from viral load data, using Markov Chain Monte Carlo methods. The data were placebo group viral load measurements collected during volunteer challenge studies, conducted by Roche, as part of the oseltamivir trials. We calculated the values of the quantities for each patient and the correlations between the quantities, symptom severity and body temperature. The greatest variation among individuals occurred in the viral load peak and area under the viral load curve. Total symptom severity correlated positively with the basic reproductive number. The most sensitive endpoint for therapeutic trials with the goal to cure patients is the duration of infection. We suggest laboratory experiments to obtain more precise estimates of virological quantities that can supplement clinical endpoint measurements. According to a 2014 report, the costs of developing a new pharmaceutical has increased by 145% since 2003. The. The10].We fitted the model to viral load data of nine patients from the placebo groups of volunteer challenge studies conducted by Roche as part of the original Oseltamivir trials, using Markov Chain Monte Carlo (MCMC) methods . All volIn addition to viral load measurements, the data also included temperature measurements and total symptom scores (Jackson score ) for eacWritten informed consent was obtained from each participant in a form approved by the institutional review boards of the University of Virginia, Charlottesville, and the University of Rochester, Rochester, NY, and subjects were compensated for participation. The review board of the named institution has approved the study in which the data we used was collected.R0 and the duration of infection , viral load AUC and peak viral load , viral load AUC and late viral decay rate , time to peak viral load and generation time , time to peak viral load and initial viral growth rate , and peak viral load and late viral decay rate . It should be noted that the infection-related quantities are related to each other via the model parameters, which explains the very high correlations.0 and total symptom score AUC , fraction of dead cells at the end of infection and duration of infection , generation time and initial viral growth rate . Here R0 and total symptom score AUC are independent, whereas the fraction of dead cells and the duration of infection, as well as the generation time and initial viral growth rate are dependent on each other via the viral dynamics model. See When we used the Benjamini-Hochberg correction, three additional correlations were significant: REven though the association is not statistically significant, we observe that Patients 3, 4 and 5 have the highest fraction of dead cells and also the highest symptom scores AUC , Fig 3.Our parameter estimates are of the same size as previous estimates from studies using comparable models (for example ). As theR0) tend to be lower than previously reported results [0 is likely to be higher. The viral strains used also differed in the various studies (influenza A Texas/36/91 (H1N1) in our study, Hong Kong/123/77 (H1N1) in [R0 values. Moreover, some of the earlier studies consider a latent infection stage which can increase estimates of R0. We did not consider a latent stage, as the data did not contain enough information to justify additional parameters.Our estimates of the basic reproductive number  in , and AlbIn our analysis, peak viral load and viral load AUC are the most variable infection-related quantities. They were also strongly correlated, indicating that viral load AUC is mainly determined by peak viral load. High variability in AUC is thought to reflect high variability in infectiousness among individuals.We found that viral load correlates significantly with body temperature and total symptom scores, but the correlations were not very strong. This may indicate that, although viral load is ultimately responsible for infection-related illness, other processes, for example the immune response, cause symptoms. Alternatively, the reporting of symptoms may be rather subjective and therefore difficult to quantify accurately.R0 has a significant positive correlation with the duration of infection and the total symptom score AUC. Consequently, R0 may be interpreted as an indicator for disease severity.We found that Viral load AUC positively correlates with peak viral load and with the late viral decay rate. Peak viral load correlates positively with the late viral decay rate. This suggests that infections with a high peak viral load tend to decline rapidly after the peak , whereas infections with a low peak viral load tend to decline more slowly .The fraction of dead cells at the end of infection correlates positively with the infection duration. This means that, according to our model, longer infections cause more severe tissue damage. In addition, we observed that the patients with the highest fraction of dead cells at the end of infection have the greatest total symptom score AUCs. A possible interpretation may be that symptoms are at least partly caused by tissue damage. The inter-individual variability in the fraction of dead cells, however, is very low , the viAs we derived in , treatmeSince patients with acute viral infections typically present at clinics after the time of peak viral load, our model suggests that it will be difficult to detect the impact of a treatment that acts on the infection rate or viral production rate. This may be one of the reasons why different studies have shown ambiguous results on the efficacy of neuraminidase inhibitors for the treatment of influenza A –7.According to our model, when treatments that act on the virus clearance rate are given before the time of peak viral load, they can slow down viral growth and reduce the viral load peak and AUC . ShortlyOur predictions strongly depend on the assumption that the infection is target-cell-limited. In infections that are not target-cell-limited and where immunity plays a greater role in reducing viral load, treatments acting on viral growth parameters are expected to have a much stronger effect after the viral load peak.The choice of endpoint in a clinical trial depends in part on the therapeutic goal. If the goal is to reduce transmission of an infectious agent, the viral load AUC, a measure of infectiousness, is a sensible endpoint. If the goal is to clear infection in a patient, other endpoints may be more useful. Based on our analyses, we make the following recommendations on clinical protocols for the assessment of treatments for acute viral infections with the goal to clear infection in a patient. Our general conclusions hold for all trial phases, but some of our suggestions concerning practical implementation may only be applicable in phase I studies.When a treatment is given before the time of peak viral load the viral load AUC is commonly used as a virological endpoint. One of the reasons for this is that the AUC is affected by the peak viral load and the duration of infection, and therefore contains more information about the course of infection than either of these quantities on their own. Consequently, the viral load AUC should be the most sensitive indicator of the efficacy of a treatment.According to our findings, however, the viral load AUC is the infection-related quantity that varies most among individuals. Due to the high natural variability among untreated patients, it should be more difficult to detect a statistically significant difference between treated and control groups. The same consideration holds for the peak viral load which is also highly variable. Conversely, the duration of infection varies much less among individuals (CV = 0.2059). Therefore, besides the reduction in AUC and peak viral load, the duration of infection should be considered as an endpoint.When a treatment is given after the peak viral load, the AUC is not a very sensitive marker of impact, because the reduction in the total area will be relatively smaller see . The incThe measured duration of infection will depend on the number of measurements taken and the sensitivity of the viral load assay. It is also easy to miss the peak viral load, if the measurement intervals are spaced too far apart. This will affect the estimate of the viral load AUC. We recommend taking at least two to three measurements per day, if possible (mainly in phase I studies). This is partly because the reduction in infection duration in treated individuals is typically not more than one day . If onlyTaking more frequent viral load measurements is essential for fitting mechanistic models of acute viral infections to data. In order to fit the model we introduced above, one needs a minimum of at least five data points taken at different times . In part50 [The lower the measurement error and the higher the sensitivity of an assay, the more accurate the data. Determining the distribution of the measurement error of a given assay can improve statistical inference. This can be achieved by running replicate measurements at each step of the assay on the same sample and from different samples taken from the same patient at one time point. In the case of influenza A, the most common assays are RT-qPCR and TCID50 ,24. InfoWe suggest a number of laboratory experiments that could help to improve estimates of model parameters and disease markers. Parameters that can be measured independently can be set to a fixed value or allow the definition of better prior distributions during the model fitting procedure, so that the remaining free parameters can be estimated with greater accuracy.For example, the decay rate of influenza A virus has been measured previously in micro-carrier culture . Viral cThe virus production rate per cell can be estimated by determining the total amount of virus produced in a culture system and dividing it by the total amount of host cells in the system . The virThese experiments may not be cheap, and may be rather complex. In order to supplement and validate data obtained from tissue culture systems, animal models may prove useful. In particular, it may be possible to obtain data on the dynamics of infected cells from serial autopsies from sacrificed animals. The more components of the virus life cycle and the human immune response can be measured independently the more complex and detailed models can be built of viral infection. Hence better assessments of therapy impact can be made. Compared to the cost of failed clinical trials, the cost and endeavour of additional laboratory experiments should be worth the attempt.S1 FileDescription of the parameter estimation procedure.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.