Nonperturbing Manner Research Articles

First Demonstration of Medical Radiation Treatment Beam Monitoring via Air Scintillation Imaging

Error rates in radiation therapy administration, although reportedly low, can have devastating consequences. Recent fatal incidents could potentially have been mitigated if real-time methods of monitoring delivery of radiation during treatment had been utilized. However, few existing methods are practical enough to be used routinely without disrupting clinical operations. The purpose of this study is to investigate a novel non-perturbing method of monitoring radiation therapy. The proposed method for monitoring radiation delivery exploits the fact that when ionizing radiation propagates through the atmosphere, it excites nitrogen gas on its path. This excitation induces weak luminescence (primarily in the 300-400 nm range) via the process of air scintillation. While not visible to the naked eye, air scintillation can be imaged with a sensitive camera to probe the physical characteristics of a radiation beam. An electron-multiplication charge-coupled device camera (f/0.95 lens) was set-up in a clinical treatment vault and was used to capture air scintillation images of kilovoltage and megavoltage beams. The vaults were prepared to block background light and a short-pass filter was utilized to block light above 440 nm. Megavoltage electron beams from a linear accelerator, as well as an orthovoltage unit (50 kVp, 30 mA) were visualized with a relatively short exposure time (10 s), showing an inverse intensity falloff (r2 = 0.89) in image intensity along the central axis. For a fixed exposure time (100 s), air scintillation was shown to be proportional to dose rate (r2 = 0.9998). As beam energy increased from 6 MeV to 20 MeV, air scintillation images exhibited a decrease in the divergence of the electron beam and improved penumbra. The irradiation of a transparent phantom also showed that Cherenkov luminescence does not interfere with the detection of air scintillation. In a final illustration of the capabilities of this new technique, air scintillation was captured during a total skin irradiation electron treatment. Air scintillation can be measured to monitor a radiation beam in an inexpensive and non-perturbing manner. This new method is particularly advantageous for monitoring a wide area in a single acquisition, which may be useful for online verification of total body/skin/lymphoid irradiation treatments. Future work on air scintillation aims to further validate this method using accurate Monte-Carlo simulations and translate this technology to the clinic.

TH-C-17A-09: Direct Visualization and Monitoring of Medical Radiation Beams in Air

Purpose: Radiation therapy errors are rare but potentially catastrophic. Recent fatal incidents could have been avoided by utilizing real-time methods of monitoring delivery of radiation during treatment. However, few existing methods are practical enough to be used routinely. The study presents the first experimental demonstration of a novel non-perturbing method of monitoring radiation therapy through the phenomena of air scintillation. Methods: Monitoring of radiation delivery was devised by leveraging the phenomena of nitrogen excitation in air by ionizing radiation. The excitation induced weak luminescence in the 300–400 nm range, a process called air scintillation. An electron-multiplication charge-coupled device camera (f/0.95 lens; 440 nm shortpass) was set-up in a clinical treatment vault and was used to capture air scintillation images of kilovoltage and megavoltage beams. Monte Carlo simulations were performed to determine the correlation of radiation dose to air scintillation. Results: Megavoltage beams from a Varian Clinac 21EX and kilovoltage beams from an orthovoltage unit (50 kVp, 30 mA) were visualized with a relatively short exposure time (10 s). Cherenkov luminescence produced in a plastic transparent phantom did not interfere with detection of air scintillation. The image intensity displayed an inverse intensity falloff (r2 = 0.89) along the central axis and was proportional to dose rate (r2 = 0.9998). As beam energy increased, the divergence of the imaged beam decreased. Last, air scintillation was visualized during a simulated total skin irradiation electron treatment. Conclusion: Air scintillation can be clinically detected to monitor a radiation beam in an inexpensive and non-perturbing manner. This new method is advantageous in monitoring for gross delivery and uniquely capable of wide area in a single acquisition, such as the case for online verification of total body / skin / lymphoid irradiation treatments.

Seeing the invisible: Direct visualization of therapeutic radiation beams using air scintillation

To assess whether air scintillation produced during standard radiation treatments can be visualized and used to monitor a beam in a nonperturbing manner. Air scintillation is caused by the excitation of nitrogen gas by ionizing radiation. This weak emission occurs predominantly in the 300-430 nm range. An electron-multiplication charge-coupled device camera, outfitted with an f/0.95 lens, was used to capture air scintillation produced by kilovoltage photon beams and megavoltage electron beams used in radiation therapy. The treatment rooms were prepared to block background light and a short-pass filter was utilized to block light above 440 nm. Air scintillation from an orthovoltage unit (50 kVp, 30 mA) was visualized with a relatively short exposure time (10 s) and showed an inverse falloff (r(2) = 0.89). Electron beams were also imaged. For a fixed exposure time (100 s), air scintillation was proportional to dose rate (r(2) = 0.9998). As energy increased, the divergence of the electron beam decreased and the penumbra improved. By irradiating a transparent phantom, the authors also showed that Cherenkov luminescence did not interfere with the detection of air scintillation. In a final illustration of the capabilities of this new technique, the authors visualized air scintillation produced during a total skin irradiation treatment. Air scintillation can be measured to monitor a radiation beam in an inexpensive and nonperturbing manner. This physical phenomenon could be useful for dosimetry of therapeutic radiation beams or for online detection of gross errors during fractionated treatments.

Analysis of the Mobilities of Band 3 Populations Associated with Ankyrin Protein and Junctional Complexes in Intact Murine Erythrocytes

Current models of the erythrocyte membrane depict three populations of band 3: (i) a population tethered to spectrin via ankyrin, (ii) a fraction attached to the spectrin-actin junctional complex via adducin, and (iii) a freely diffusing population. Because many studies of band 3 diffusion also distinguish three populations of the polypeptide, it has been speculated that the three populations envisioned in membrane models correspond to the three fractions observed in diffusion analyses. To test this hypothesis, we characterized band 3 diffusion by single-particle tracking in wild-type and ankyrin- and adducin-deficient erythrocytes. We report that ∼40% of total band 3 in wild-type murine erythrocytes is attached to ankyrin, whereas ∼33% is immobilized by adducin, and ∼27% is not attached to any cytoskeletal anchor. More detailed analyses reveal that mobilities of individual ankyrin- and adducin-tethered band 3 molecules are heterogeneous, varying by nearly 2 orders of magnitude and that there is considerable overlap in diffusion coefficients for adducin and ankyrin-tethered populations. Taken together, the data suggest that although the ankyrin- and adducin-immobilized band 3 can be monitored separately, significant heterogeneity still exists within each population, suggesting that structural and compositional properties likely vary considerably within each band 3 complex.

Measurement of genome-wide RNA synthesis and decay rates with Dynamic Transcriptome Analysis (DTA)

Standard transcriptomics measures total cellular RNA levels. Our understanding of gene regulation would be greatly improved if we could measure RNA synthesis and decay rates on a genome-wide level. To that end, the Dynamic Transcriptome Analysis (DTA) method has been developed. DTA combines metabolic RNA labeling with standard transcriptomics to measure RNA synthesis and decay rates in a precise and non-perturbing manner. Here, we present the open source R/Bioconductor software package DTA. It implements all required bioinformatics steps that allow the accurate absolute quantification and comparison of RNA turnover.

Discrete States of a Protein Interaction Network Govern Interphase and Mitotic Microtubule Dynamics

The cytoplasm of eukaryotic cells is thought to adopt discrete “states” corresponding to different steady states of protein networks that govern changes in subcellular organization. For example, in Xenopus eggs, the interphase to mitosis transition is induced solely by activation of cyclin-dependent kinase 1 (CDK1) that phosphorylates many proteins leading to a reorganization of the nucleus and assembly of the mitotic spindle. Among these changes, the large array of stable microtubules that exists in interphase is replaced by short, highly dynamic microtubules in metaphase. Using a new visual immunoprecipitation assay that quantifies pairwise protein interactions in a non-perturbing manner in Xenopus egg extracts, we reveal the existence of a network of interactions between a series of microtubule-associated proteins (MAPs). In interphase, tubulin interacts with XMAP215, which is itself interacting with XKCM1, which connects to APC, EB1, and CLIP170. In mitosis, tubulin interacts with XMAP215, which is connected to EB1. We show that in interphase, microtubules are stable because the catastrophe-promoting activity of XKCM1 is inhibited by its interactions with the other MAPs. In mitosis, microtubules are short and dynamic because XKCM1 is free and has a strong destabilizing activity. In this case, the interaction of XMAP215 with EB1 is required to counteract the strong activity of XKCM1. This provides the beginning of a biochemical description of the notion of “cytoplasmic states” regarding the microtubule system.

Synergistic Transmembrane Alignment of the Antimicrobial Heterodimer PGLa/Magainin

The antimicrobial activity of amphipathic alpha-helical peptides is usually attributed to the formation of pores in bacterial membranes, but direct structural information about such a membrane-bound state is sparse. Solid state (2)H-NMR has previously shown that the antimicrobial peptide PGLa undergoes a concentration-dependent realignment from a surface-bound S-state to a tilted T-state. The corresponding change in helix tilt angle from 98 to 125 degrees was interpreted as the formation of PGLa/magainin heterodimers residing on the bilayer surface. Under no conditions so far, has an upright membrane-inserted I-state been observed in which a transmembrane helix alignment would be expected. Here, we have demonstrated that PGLa is able to assume such an I-state in a 1:1 mixture with magainin 2 at a peptide-to-lipid ratio as low as 1:100 in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol model membranes. This (2)H-NMR analysis is based on seven orientational constraints from Ala-3,3,3-d(3) substituted in a non-perturbing manner for four native Ala residues as well as two Ile and one Gly. The observed helix tilt of 158 degrees is rationalized by the formation of heterodimers. This structurally synergistic effect between the two related peptides from the skin of Xenopus laevis correlates very well with their known functional synergistic mode of action. To our knowledge, this example of PGLa is the first case where an alpha-helical antimicrobial peptide is directly shown to assume a transmembrane state that is compatible with the postulated toroidal wormhole pore structure.

Spectroscopic translation of cell–material interactions

The characterization of cellular interactions with a biomaterial surface is important to the development of novel biomaterials. Traditional methods used to characterize processes such as cellular adhesion and differentiation on biomaterials can be time consuming, and destructive, and are not amenable to quantitative assessment in situ. As the development of novel biomaterials shifts towards small-scale, combinatorial, and high throughput approaches, new techniques will be required to rapidly screen and characterize cell/biomaterial interactions. Towards this goal, we assessed the feasibility of using 4-dimensional elastic light-scattering fingerprinting (4D-ELF) to describe the differentiation of human aortic smooth muscle cells (HASMCs), as well as the adhesion, and apoptotic processes of human aortic endothelial cells (HAECs), in a quantitative and non-perturbing manner. HASMC and HAEC were cultured under conditions to induce cell differentiation, attachment, and apoptosis which were evaluated via immunohistochemistry, microscopy, biochemistry, and 4D-ELF. The results show that 4D-ELF detected changes in the size distributions of subcellular organelles and structures that were associated with these specific cellular processes. 4D-ELF is a novel way to assess cell phenotype, strength of adhesion, and the onset of apoptosis on a biomaterial surface and could potentially be used as a rapid and quantitative screening tool to provide a more in-depth understanding of cell/biomaterial interactions.

Characterization of Freshwater Natural Aquatic Colloids by Atomic Force Microscopy (AFM)

Atomic force microscopy (AFM) has been used to image and quantify riverine colloids in a quantitative and relatively nonperturbing manner. Three main classes of material have been imaged including fibrils (about 10 nm in diameter and 100 nm or more in length), discrete, near-spherical, small colloids primarily below 30-50 nm in diameter, and a surface film, of at least several nanometers thickness, which coatsthe entire mica surface within 30 min of exposure to river water. Colloid structure was found to vary as a function of pH, particularly at high pH. Substantially different structures were observed at high pH values, with the loss of the near-spherical colloids possibly due to rearrangement and aggregation. In addition,film thicknesses of up to 100 nm were estimated on the silicon nitride AFM cantilever after 30 h of deposition in the same water (unperturbed and size fractionated). The observation of these surface films has important implications for understanding the mechanisms by which colloids might bind trace elements. In particular, development of surface coatings implies that binding of pollutants (at least initial surface binding) may be dominated by adsorbed surface layers.

Correlation reflectometry: An experimental investigation of measurement capability

The correlation reflectometer was conceived as a method of determining the interior correlation length of plasma turbulence in a nonperturbing and localized manner. Currently, measurements on different machines have produced different lengths and tendencies. Theories have been used to interpret the data from this diagnostic, but the diagnostic and theories remain unverified by direct validation with other diagnostics. As part of its ongoing research program aimed at the understanding and full utilization of reflectometry, UCLA has initiated a direct comparison between turbulent correlation lengths as measured by reflectometry and Langmuir probes. This investigation is performed on the CCT tokamak at UCLA. Initial results show good agreement between the two diagnostics, with radial coherence lengths in the range 1.0–3.0 cm depending upon the frequency of the fluctuation. A detailed description of the experiment and initial results are presented.

Titration calorimetry as a binding assay for lipid-binding proteins.

Titration calorimetry has been evaluated as a method for obtaining binding constants and thermodynamic parameters for the cytosolic fatty acid- and lipid-binding proteins. An important feature of this method was its ability to accurately determine binding constants in a non-perturbing manner. The equilibrium was not perturbed, since there was no requirement to separate bound and free ligand in order to obtain binding parameters. Also, the structure of the lipid-protein complex was not perturbed, since native ligands were used rather than non-native analogues. As illustrated for liver fatty acid-binding protein, the method distinguished affinity classes whose dissociation constants differed by an order of magnitude or less. It also distinguished endothermic from exothermic binding reactions, as illustrated for the binding of two closely related bile salts to ileal lipid-binding protein. The main limitations of the method were its relatively low sensitivity and the difficulty working with highly insoluble ligands, such as cholesterol or saturated long-chain fatty acids. However, the signal-to-noise ratio was improved by manipulating the buffer conditions, as illustrated for oleate binding to rat intestinal fatty acid binding protein. Binding parameters are reported for oleate interactions with several wild-type and mutant lipid-binding proteins from intestine. Where possible, the binding parameters obtained from calorimetry were compared with results obtained from fluorescence and Lipidex binding assays of comparable systems.

Diagnostic system for determining the internal structure of a field reversed configuration

A set of diagnostics has been developed to determine the structure and internal density profile of a field reversed configuration (FRC) in a nonperturbing manner. The primary diagnostic is a 50-channel tomographic array which uses bremsstrahlung radiation from the FRC in either the visible or near infrared. End-on imaging in both the visible and soft x-ray regions are used to improve inversion accuracy by providing additional information in the form of constraints. A five-channel interferometer will be used as a baseline check on the imaging diagnostic, and provide information on the basic symmetry of the FRC. Up to 80 BΘ probes, with the application of a diagnostic axial current, will be used to detect external changes in separatrix shape. Comparison of diagnostic signals with signals calculated from 3-D MHD simulations will provide for additional determination of internal mode activity.

Observations of spheromak equilibria which differ from the minimum-energy state and have internal kink distortions.

Experimental spheromak magnetic equilibria are measured which differ significantly from the minimum-energy state, and are well described by a numerical model where ${j}_{?}$/B has a linear dependence on the poloidal flux function. Equilibria are determined in a nonperturbing manner by the combination of measurements of flux-conserver image currents with calculations from this model. These equilibria are corroborated by the observation of nondisruptive rotating internal kink distortions (with toroidal mode numbers n=1, 2, and 3), coupled with theoretical thresholds for the onset of these modes.

Spectroscopic ellipsometry: A new tool for nondestructive depth profiling and characterization of interfaces

The ability of spectroscopic ellipsometry to analyze the multilayer surface structure of an implanted semiconductor with 1–2 Å resolution has been demonstrated. Spectroscopic ellipsometric data from 1.5 to 4.5 eV on a number of self-ion-implanted silicon samples have been analyzed using the regression analysis technique. It is shown that such a procedure can yield information nondestructively and in a nonperturbing manner: (i) on the depth profile of multilayer structures; (ii) quantitative information on the thicknesses of each layer (within 90% confidence limits); (iii) the structure (whether crystalline or amorphous) as well as the degree of crystallinity in the region; (iv) characterize the oxide layer if present on specimen; and (v) microroughness of the surface, if present. Furthermore, it is shown that these results are in excellent quantitative agreement with the completely independent results obtained on the same specimens using cross-section transmission electron microscopy.

Nondestructive depth profiling by spectroscopic ellipsometry

It is shown that spectroscopic ellipsometry (SE) studies followed by the regression analysis of the SE data can yield information nondestructively and in a nonperturbing manner on the depth profile of multilayer structure; such as (i) quantitative information on the thickness and the dielectric function of each layer, (ii) the structure (whether crystalline or amorphous) as well as the degree of crystallinity of each layer, (iii) characterization of the oxide layer if present on the specimen, and (iv) microroughness of the surface, if present. Furthermore, it is shown that these results are in excellent agreement with the results obtained on the same specimens using cross-section transmission electron microscopy.

Use of laser-generated acoustic pulses to measure the electric field inside a solid dielectric

We describe a technique for measuring the electric field inside solid dielectrics and apply it to a polymethylmethacrylate sample. The technique uses a laser-generated acoustic pulse to deform the sample inhomogeneously in a nondestructive, nonperturbing manner. From the electrical response of the sample during deformation, the macroscopic electric field as a function of position may be calculated. We achieved a sensitivity of 5×104 V/m and a spatial resolution of 50 μm in a 3-mm-thick sample.