Thoracic target volume delineation using various maximum-intensity projection computed tomography image sets for stereotactic body radiation therapy

The motion of lung tumors during respiration makes the accurate delivery of radiation therapy to the thorax difficult because it increases the uncertainty of target position. The adoption of four-dimensional computed tomography (4D-CT) has allowed us to determine how a tumor moves with respiration for each individual patient. Using information acquired during a 4D-CT scan, we can define the target, visualize motion, and calculate dose during the planning phase of the radiotherapy process. One image data set that can be created from the 4D-CT acquisition is the maximum-intensity projection (MIP). The MIP can be used as a starting point to define the volume that encompasses the motion envelope of the moving gross target volume (GTV). Because of the close relationship that exists between the MIP and the final target volume, we investigated four MIP data sets created with different methodologies (3 using various 4D-CT sorting implementations, and one using all available cine CT images) to compare target delineation. It has been observed that changing the 4D-CT sorting method will lead to the selection of a different collection of images; however, the clinical implications of changing the constituent images on the resultant MIP data set are not clear. There has not been a comprehensive study that compares target delineation based on different 4D-CT sorting methodologies in a patient population. We selected a collection of patients who had previously undergone thoracic 4D-CT scans at our institution, and who had lung tumors that moved at least 1 cm. We then generated the four MIP data sets and automatically contoured the target volumes. In doing so, we identified cases in which the MIP generated from a 4D-CT sorting process under-represented the motion envelope of the target volume by more than 10% than when measured on the MIP generated from all of the cine CT images. The 4D-CT methods suffered from duplicate image selection and might not choose maximum extent images. Based on our results, we suggest utilization of a MIP generated from the full cine CT data set to ensure a representative inclusive tumor extent, and to avoid geometric miss.

Intact flagellar motor of Borrelia burgdorferi revealed by cryo-electron tomography: evidence for stator ring curvature and rotor/C-ring assembly flexion.

The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact Borrelia burgdorferi to produce a three-dimensional (3-D) model of the in situ flagellar motor without imposing rotational symmetry. Structural details of B. burgdorferi, including a layer of outer surface proteins, were clearly visible in the resulting 3-D reconstructions. By averaging the 3-D images of approximately 1,280 flagellar motors, a approximately 3.5-nm-resolution model of the stator and rotor structures was obtained. flgI transposon mutants lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Treatment of intact organisms with the nonionic detergent NP-40 resulted in dissolution of the outermost portion of the motor structure and the C ring, providing insight into the in situ arrangement of the stator and rotor structures. Structural elements associated with the stator followed the curvature of the cytoplasmic membrane. The rotor and the C ring also exhibited angular flexion, resulting in a slight narrowing of both structures in the direction perpendicular to the cell axis. These results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C-ring assembly during flagellar rotation.

3D visualization of HIV virions by cryoelectron tomography.

The structure of the human immunodeficiency virus (HIV) and some of its components have been difficult to study in three-dimensions (3D) primarily because of their intrinsic structural variability. Recent advances in cryoelectron tomography (cryo-ET) have provided a new approach for determining the 3D structures of the intact virus, the HIV capsid, and the envelope glycoproteins located on the viral surface. A number of cryo-ET procedures related to specimen preservation, data collection, and image processing are presented in this chapter. The techniques described herein are well suited for determining the ultrastructure of bacterial and viral pathogens and their associated molecular machines in situ at nanometer resolution.

Cryo-electron tomography of Kaposi's sarcoma-associated herpesvirus capsids reveals dynamic scaffolding structures essential to capsid assembly and maturation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a recently discovered DNA tumor virus that belongs to the gamma-herpesvirus subfamily. Though numerous studies on KSHV and other herpesviruses, in general, have revealed much about their multilayered organization and capsid structure, the herpesvirus capsid assembly and maturation pathway remains poorly understood. Structural variability or irregularity of the capsid internal scaffolding core and the lack of adequate tools to study such structures have presented major hurdles to earlier investigations employing more traditional cryo-electron microscopy (cryoEM) single particle reconstruction. In this study, we used cryo-electron tomography (cryoET) to obtain 3D reconstructions of individual KSHV capsids, allowing direct visualization of the capsid internal structures and systematic comparison of the scaffolding cores for the first time. We show that B-capsids are not a structurally homogenous group; rather, they represent an ensemble of "B-capsid-like" particles whose inner scaffolding is highly variable, possibly representing different intermediates existing during the KSHV capsid assembly and maturation. This information, taken together with previous observations, has allowed us to propose a detailed pathway of herpesvirus capsid assembly and maturation.

Unique structures in a tumor herpesvirus revealed by cryo-electron tomography and microscopy.

Gammaherpesviruses, including the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, are causative agents of lymphomas and other malignancies. The structural characterization of these viruses has been limited due to difficulties in obtaining adequate amount of virion particles. Here we report the first three-dimensional structural characterization of a whole gammaherpesvirus virion by an emerging integrated approach of cryo-electron tomography combined with single-particle cryo-electron microscopy, using murine gammaherpesvirus-68 (MHV-68) as a model system. We found that the MHV-68 virion consists of distinctive envelope and tegument compartments, and a highly conserved nucleocapsid. Two layers of tegument are identified: an inner tegument layer tethered to the underlying capsid and an outer, flexible tegument layer conforming to the overlying, pleomorphic envelope, consistent with the sequential viral tegumentation process inside host cells. Surprisingly, comparison of the MHV-68 virion and capsid reconstructions shows that the interactions between the capsid and inner tegument proteins are completely different from those observed in alpha and betaherpesviruses. These observations support the notion that the inner layer tegument across different subfamilies of herpesviruses has evolved significantly to confer specific characteristics related to viral-host interactions, in contrast to a highly conserved capsid for genome encapsidation and protection.

Direct visualization of the putative portal in the Kaposi's sarcoma-associated herpesvirus capsid by cryoelectron tomography.

Genetic and biochemical studies have suggested the existence of a bacteriophage-like, DNA-packaging/ejecting portal complex in herpesviruses capsids, but its arrangement remained unknown. Here, we report the first visualization of a unique vertex in the Kaposi's sarcoma-associated herpesvirus (KSHV) capsid by cryoelectron tomography, thus providing direct structural evidence for the existence of a portal complex in a gammaherpesvirus. This putative KSHV portal is an internally localized, umbilicated structure and lacks all of the external machineries characteristic of portals in DNA bacteriophages.

DUAL ENERGY COMPUTED TOMOGRAPHY FOR PROTON THERAPY TREATMENT PLANNING

Proton radiation therapy is gaining popularity because of the unique characteristics of its dose distribution, e.g., high dose-gradient at the distal end of the percentage-depth-dose curve (known as the Bragg peak). The high dose-gradient offers the possibility of delivering high dose to the target while still sparing critical organs distal to the target. However, the high dose-gradient is a double-edged sword: a small shift of the highly conformal high-dose area can cause the target to be substantially under-dosed or the critical organs to be substantially over-dosed. Because of that, large margins are required in treatment planning to ensure adequate dose coverage of the target, which prevents us from realizing the full potential of proton beams. Therefore, it is critical to reduce uncertainties in the proton radiation therapy. One major uncertainty in a proton treatment is the range uncertainty related to the estimation of proton stopping power ratio (SPR) distribution inside a patient. The SPR distribution inside a patient is required to account for tissue heterogeneities when calculating dose distribution inside the patient. In current clinical practice, the SPR distribution inside a patient is estimated from the patient’s treatment planning computed tomography (CT) images based on the CT number-to-SPR calibration curve. The SPR derived from a single CT number carries large uncertainties in the presence of human tissue composition variations, which is the major drawback of the current SPR estimation method. We propose to solve this problem by using dual energy CT (DECT) and hypothesize that the range uncertainty can be reduced by a factor of two from currently used value of 3.5%. A MATLAB program was developed to calculate the electron density ratio (EDR) and effective atomic number (EAN) from two CT measurements of the same object. An empirical relationship was discovered between mean excitation energies and EANs existing in human body tissues. With the MATLAB program and the empirical relationship, a DECT-based method was successfully developed to derive SPRs for human body tissues (the DECT method). The DECT method is more robust against the uncertainties in human tissues compositions than the current single-CT-based method, because the DECT method incorporated both density and elemental composition information in the SPR estimation. Furthermore, we studied practical limitations of the DECT method. We found that the accuracy of the DECT method using conventional kV-kV x-ray pair is susceptible to CT number variations, which compromises the theoretical advantage of the DECT method. Our solution to this problem is to use a different x-ray pair for the DECT. The accuracy of the DECT method using different combinations of x-ray energies, i.e., the kV-kV, kV-MV and MV-MV pair, was compared using the measured imaging uncertainties for each case. The kV-MV DECT was found to be the most robust against CT number variations. In addition, we studied how uncertainties propagate through the DECT calculation, and found general principles of selecting x-ray pairs for the DECT method to minimize its sensitivity to CT number variations. The uncertainties in SPRs estimated using the kV-MV DECT were analyzed further and compared to those using the stoichiometric method. The uncertainties in SPR estimation can be divided into five categories according to their origins: the inherent uncertainty, the DECT modeling uncertainty, the CT imaging uncertainty, the uncertainty in the mean excitation energy, and SPR variation with proton energy. Additionally, human body tissues were divided into three tissue groups – low density (lung) tissues, soft tissues and bone tissues. The uncertainties were estimated separately because their uncertainties were different under each condition. An estimate of the composite range uncertainty (2s) was determined for three tumor sites – prostate, lung, and head-and-neck, by combining the uncertainty estimates of all three tissue groups, weighted by their proportions along typical beam path for each treatment site. In conclusion, the DECT method holds theoretical advantages in estimating SPRs for human tissues over the current single-CT-based method. Using existing imaging techniques, the kV-MV DECT approach was capable of reducing the range uncertainty from the currently used value of 3.5% to 1.9%-2.3%, but it is short to reach our original goal of reducing the range uncertainty by a factor of two. The dominant source of uncertainties in the kV-MV DECT was the uncertainties in CT imaging, especially in MV CT imaging. Further reduction in beam hardening effect, the impact of scatter, out-of-field object etc. would reduce the Hounsfeld Unit variations in CT imaging. The kV-MV DECT still has the potential to reduce the range uncertainty further.

A convolution model for energy transport in a therapeutic fast neutron beam

A three-dimensional model has been proposed that uses Monte Carlo and fast Fourier transform convolution techniques to calculate the dose distribution from a fast neutron beam. This method transports scattered neutrons and photons in the forward, lateral, and backward directions and protons, electrons, and positrons in the forward and lateral directions by convolving energy spread kernels with initial interaction available energy distributions. The primary neutron and photon spectrums have been derived from narrow beam attenuation measurements. The positions and strengths of the effective primary neutron, scattered neutron, and photon sources have been derived from dual ion chamber measurements. The size of the effective primary neutron source has been measured using a copper activation technique. Heterogeneous tissue calculations require a weighted sum of two convolutions for each component since the kernels must be invariant for FFT convolution. Comparisons between calculations and measurements were performed for several water and heterogeneous phantom geometries. ^

THE CALIBRATION AND IMPLEMENTATION OF CR-39 NUCLEAR TRACK DETECTOR MATERIAL AS A FAST NEUTRON MICRODOSIMETER

A detailed microdosimetric characterization of the M. D. Anderson 42 MeV (p,Be) fast neutron beam was performed using the techniques of microdosimetry and a 1/2 inch diameter Rossi proportional counter. These measurements were performed at 5, 15, and 30 cm depths on the central axis, 3 cm inside, and 3 cm outside the field edge for 10 $\times$ 10 and 20 $\times$ 20 cm field sizes. Spectra were also measured at 5 and 15 cm depth on central axis for a 6 $\times$ 6 cm field size. Continuous slowing down approximation calculations were performed to model the nuclear processes that occur in the fast neutron beam. Irradiation of the CR-39 was performed using a tandem electrostatic accelerator for protons of 10, 6, and 3 MeV and alpha particles of 15, 10, and 7 MeV incident energy on target at angles of incidence from 0 to 85 degrees. The critical angle as well as track etch rate and normal incidence diameter versus linear energy transfer (LET) were obtained from these measurements. The bulk etch rate was also calculated from these measurements. Dose response of the material was studied, and the angular distribution of charged particles created by the fast neutron beam was measured with CR-39. The efficiency of CR-39 was calculated versus that of the Rossi chamber, and an algorithm was devised for derivation of LET spectra from the major and minor axis dimensions of the observed tracks. The CR-39 was irradiated in the same positions as the Rossi chamber, and the derived spectra were compared directly. ^