Label-free detection of single biological molecules using microtoroid optical resonators

Being able to detect a single molecule without the use of labels has been a long standing goal of bioengineers and physicists. This would simplify applications ranging from single molecular binding studies to those involving public health and security, improved drug screening, medical diagnostics, and genome sequencing. One promising technique that has the potential to detect single molecules is the microtoroid optical resonator. The main obstacle to detecting single molecules, however, is decreasing the noise level of the measurements such that a single molecule can be distinguished from background. We have used laser frequency locking in combination with balanced detection and data processing techniques to reduce the noise level of these devices and report the detection of a wide range of nanoscale objects ranging from nanoparticles with radii from 100 to 2.5 nm, to exosomes, ribosomes, and single protein molecules (mouse immunoglobulin G and human interleukin-2). We further extend the exosome results towards creating a non-invasive tumor biopsy assay. Our results, covering several orders of magnitude of particle radius (100 nm to 2 nm), agree with the `reactive' model prediction for the frequency shift of the resonator upon particle binding. In addition, we demonstrate that molecular weight may be estimated from the frequency shift through a simple formula, thus providing a basis for an ``optical mass spectrometer'' in solution. We anticipate that our results will enable many applications, including more sensitive medical diagnostics and fundamental studies of single receptor-ligand and protein-protein interactions in real time. The thesis summarizes what we have achieved thus far and shows that the goal of detecting a single molecule without the use of labels can now be realized.

I. The 3.7 Å crystal structure of horse heart ferricytochrome C. II. The application of the Karle-Hauptman tangent formula to protein phasing

I. The 3.7 Å Crystal Structure of Horse Heart Ferricytochrome C.

The crystal structure of horse heart ferricytochrome c has been determined to a resolution of 3.7 Å using the multiple isomorphous replacement technique. Two isomorphous derivatives were used in the analysis, leading to a map with a mean figure of merit of 0.458. The quality of the resulting map was extremely high, even though the derivative data did not appear to be of high quality.

Although it was impossible to fit the known amino acid sequence to the calculated structure in an unambiguous way, many important features of the molecule could still be determined from the 3.7 Å electron density map. Among these was the fact that cytochrome c contains little or no α-helix. The polypeptide chain appears to be wound about the heme group in such a way as to form a loosely packed hydrophobic core in the molecule.

The heme group is located in a cleft on the molecule with one edge exposed to the solvent. The fifth coordinating ligand is His 18 and the sixth coordinating ligand is probably neither His 26 nor His 33.

The high resolution analysis of cytochrome c is now in progress and should be completed within the next year.

II. The Application of the Karle-Hauptman Tangent Formula to Protein Phasing.

The Karle-Hauptman tangent formula has been shown to be applicable to the refinement of previously determined protein phases. Tests were made with both the cytochrome c data from Part I and a theoretical structure based on the myoglobin molecule. The refinement process was found to be highly dependent upon the manner in which the tangent formula was applied. Iterative procedures did not work well, at least at low resolution.

The tangent formula worked very well in selecting the true phase from the two possible phase choices resulting from a single isomorphous replacement phase analysis. The only restriction on this application is that the heavy atoms form a non-centric cluster in the unit cell.

Pages 156 through 284 in this Thesis consist of previously published papers relating to the above two sections. References to these papers can be found on page 155.

Space growth studies of Ce-doped Bi12SiO20 single crystal

Ce-doped Bi12SiO20 (BSO) single crystal was grown on board of the Chinese Spacecraft-Shenzhou No. 3. A cylindrical crystal, 10 mm in diameter and 40 mm in length, was obtained. The morphology of crystals is significantly different for ground- and space-grown portions. The space- and ground-grown crystals have been characterized by Cc concentration distribution, X-ray rocking curve absorption spectrum and micro-Raman spectrum. The results show that the quality of Ce-doped BSO crystal grown in space is more homogeneous and more perfect than that of ground grown one. (C) 2004 Published by Elsevier B.V.

Single-mode condition for silicon rib waveguides with large cross sections

The single-mode condition for silicon rib waveguides with large cross sections is obtained using a numerical method based on the finite-difference beam propagation method (FD-BPM). An ultrawide computation window is used to contain the wide mode profile near cutoff. Comparison with previous results shows that the formula predicted by the mode-matching technique is in a better agreement with our results. (C) 2004 Society of Photo-Optical Instrumentation Engineers.

Comparison of space- and ground-grown Ce: Bi12SiO20 single crystals

Ce doped Bi12SiO20 single crystals were grown either on board of the Chinese Spacecraft-Shenzhou No.3 (SZ-3) or on the ground at the same conditions with the exception of microgravity. The surface morphology of crystals clearly showed significant differences between the space- and ground-grown portions. The space- and ground-grown crystals have been measured by X-ray rocking curve, Cc concentration distribution in growth direction, dislocation density, absorption spectrums. These results show that the compositional homogeneity and structural perfection of Ce doped crystal grown in space are obviously improved.

Simple method for estimating minimum bend radii of SOI single mode curved rib waveguides

A simple method based on the effective index method was used to estimate the minimum bend radii of curved SOI waveguides. An analytical formula was obtained to estimate the minimum radius of curvature at which the mode becomes cut off due to the side radiative loss.

Multiplicity factor and diffraction geometry factor for single crystal X-ray diffraction analysis and measurement of phase content in cubic GaN/GaAs(001) epilayers

On the basis of integrated intensity of rocking curves, the multiplicity factor and the diffraction geometry factor for single crystal X-ray diffraction (XRD) analysis were proposed and a general formula for calculating the content of mixed phases was obtained. With a multifunction four-circle X-ray double-crystal diffractometer, pole figures of cubic (002), {111} and hexagonal {1010} and reciprocal space mapping were measured to investigate the distributive character of mixed phases and to obtain their multiplicity factors and diffraction geometry factors. The contents of cubic twins and hexagonal inclusions were calculated by the integrated intensities of rocking curves of cubic (002), cubic twin {111}, hexagonal {1010} and {1011}.

Monostable-Bistable Transition Logic Element (MOBILE) Model for Single-Electron Transistors

The traditional monostable-bistable transition logic element (MOBILE) structure is usually composed of resonant tunneling diodes (RTD). This letter describes a new type MOBILE structure consisting of single-electron transistors (i.e. SET-MOBILE). The analytical model of single-electron transistors ( SET) has been considered three states (including an excited state) of the discrete quantum energy levels. The simulation results show negative differential conductance (NDC) characteristics in I-DS-V-DS curve. The SET-MOBILE utilizing NDC characteristics can successfully realize the basic logic functions as the RTD-MOBILE.

Structural optimizations of SOI-based single-mode rib waveguide bends

Effects of structure parameters on bend loss of rib silicon-on-insulator (Sol) bend waveguides have been analyzed by means of effective index method (EIM) and 2D bend loss formula. The simulation results indicate that the bend loss decreases with the increase of bend radius and waveguide width, as well as with the decrease of the step factor of the rib waveguide. Moreover, the optional structure parameters have been found when bend waveguides are single-mode.

Single particle properties in asymmetric nuclear matter and TBF rearrangement effect

We have developed the formula and the numerical code for calculating the rearrangement contribution to the single particle (s.p.) properties in asymmetric nuclear matter induced by three-body forces within the framework of the Brueckner theory extended to include a microscopic three-body force (TBF). We have investigated systematically the TBF-induced rearrangement effect on the s.p. properties and their isospin-behavior in neutron-rich nuclear medium. It is shown that the TBF induces a repulsive rearrangement contribution to the s.p. potential in nuclear medium. The repulsion of the TBF rearrangement contribution increases rapidly as a function of density and nucleon momentum. It reduces largely the attraction of the BHF s.p. potential and enhances strongly the momentum dependence of the s.p. potential at large densities and high-momenta. The TBF rearrangement effect on symmetry potential is to enhances its repulsion (attraction) on neutrons (protons) in dense asymmetric nuclear matter.

Structural diversity of lanthanide-amino acid complexes under near physiological pH conditions and their recognition of single-stranded DNA

We reported here four structures of lanthanide-amino acid complexes obtained under near physiological pH conditions and their individual formula can be described as [Tb-2(DL-Cys)(4)(H2O)(8)]Cl-2 (1), [Eu-4(mu(3)-OH)(4)(L-Asp)(2)(L-HAsp)(3)(H2O)(7)] Cl center dot 11.5H(2)O (2), [Eu-8-(L-HVal) (16)(H2O)(32)]Cl-24 center dot 12.5H(2)O (3), and [Tb-2(DL-HVal)(4)(H2O)(8)]Cl-6 center dot 2H(2)O (4). These complexes showed diverse structures and have shown potential application in DNA detection. We studied the interactions of the complexes with five single-stranded DNA and found different fluorescence enhancement, binding affinity and binding stoichiometry when the complexes are bound to DNA.

Hydrothermal synthesis and X-ray single crystal structure of bimetallic cluster complex [{Co(phen)(2)}(2)V4O12]center dot H2O

A new bimetallic cluster complex with the formula [{Co(phen)(2)}(2)V4O12](H2O)-H-. was synthesized from the hydrothermal reaction of V2O5, H2C2O4, Co(NO3)(2), 1,10-phenanthroline (phen), (C4H9)(4)NOH and water. The compound crystallizes in an orthorhombic system with space group Pbcn and unit cell parameters a = 19.106(3) Angstrom, b = 15.250(3) Angstrom, c = 16.321(2) Angstrom, V = 4755.4(13) Angstrom(3), Z = 4 and R = 0.0318. The bimetallic cluster complex [{Co(phen)(2)}(2)V4O12](H2O)-H-. is composed of a discrete V4O124- cluster eovalently attached to two [Co(phen)(2)](2+) fragments and the discrete hexanuclear bimetallic clusters of [{Co(phen)(2)}(V4O12)-V-2](H2O)-H-. are further extended into interesting three-dimensional supermolecular arrays via pi-pi stacking interactions of phen groups. Other characterizations by elemental analysis, IR, and thermal analysis are also described.

Estimation of individual phases from the four-phase structure invariants in the single isomorphous replacement case

The probability distribution of the four-phase invariants in the case of single isomorphous replacement has been developed to estimate some individual phases. An example of its application to obtain the phases having special values of 0, pi or +/-pi /2 is given for a known protein structure in space group P2(1)2(1)2(1). The phasing procedure includes the determination of starting phases and an iterative calculation. The initial values of starting phases, which are required by the formula, can be obtained from the estimate of one-phase seminvariants and by specifying the origin and enantiomorph. In addition, the calculations lead to two sets of possible phases for each type of reflection by assigning arbitrarily an initial phase value. The present method provides a possibility for the multisolution technique to increase greatly the number of known phases while keeping the number of the trials quite small.

Ship-board measurements and estimations of air-sea fluxes in the western tropical Pacific during TOGA COARE

Direct air-sea flux measurements were made on RN Kexue #1 at 40 degrees S, 156 degrees E during the Tropical Ocean Global Atmosphere (TOGA) Coupled Ocean-Atmospheric Response Experiment (COARE) Intensive Observation Period (IOP). An array of six accelerometers was used to measure the motion of the anchored ship, and a sonic anemometer and Lyman-alpha hygrometer were used to measure the turbulent wind vector and specific humidity. The contamination of the turbulent wind components by ship motion was largely removed by an improvement of a procedure due to Shao based on the acceleration signals. The scheme of the wind correction for ship motion is briefly outlined. Results are presented from data for the best wind direction relative to the ship to minimize flow distortion effects. Both the time series and the power spectra of the sonic-measured wind components show swell-induced ship motion contamination, which is largely removed by the accelerometer correction scheme, There was less contamination in the longitudinal wind component than in the vertical and transverse components. The spectral characteristics of the surface-layer turbulence properties are compared with those from previous land and ocean results, Momentum and latent heat fluxes were calculated by eddy correlation and compared to those estimated by the inertial dissipation method and the TOGA COARE bulk formula. The estimations of wind stress determined by eddy correlation are smaller than those from the TOGA COARE bulk formula, especially for higher wind speeds, while those from the bulk formula and inertial dissipation technique are generally in agreement. The estimations of latent heal flux from the three different methods are in reasonable agreement. The effect of the correction for ship motion on latent heat fluxes is not as large as on momentum fluxes.

On-board Robotic Multi-pinhole SPECT System for Region-of-interest (ROI) Imaging

On-board image guidance, such as cone-beam CT (CBCT) and kV/MV 2D imaging, is essential in many radiation therapy procedures, such as intensity modulated radiotherapy (IMRT) and stereotactic body radiation therapy (SBRT). These imaging techniques provide predominantly anatomical information for treatment planning and target localization. Recently, studies have shown that treatment planning based on functional and molecular information about the tumor and surrounding tissue could potentially improve the effectiveness of radiation therapy. However, current on-board imaging systems are limited in their functional and molecular imaging capability. Single Photon Emission Computed Tomography (SPECT) is a candidate to achieve on-board functional and molecular imaging. Traditional SPECT systems typically take 20 minutes or more for a scan, which is too long for on-board imaging. A robotic multi-pinhole SPECT system was proposed in this dissertation to provide shorter imaging time by using a robotic arm to maneuver the multi-pinhole SPECT system around the patient in position for radiation therapy.

A 49-pinhole collimated SPECT detector and its shielding were designed and simulated in this work using the computer-aided design (CAD) software. The trajectories of robotic arm about the patient, treatment table and gantry in the radiation therapy room and several detector assemblies such as parallel holes, single pinhole and 49 pinholes collimated detector were investigated. The rail mounted system was designed to enable a full range of detector positions and orientations to various crucial treatment sites including head and torso, while avoiding collision with linear accelerator (LINAC), patient table and patient.

An alignment method was developed in this work to calibrate the on-board robotic SPECT to the LINAC coordinate frame and to the coordinate frames of other on-board imaging systems such as CBCT. This alignment method utilizes line sources and one pinhole projection of these line sources. The model consists of multiple alignment parameters which maps line sources in 3-dimensional (3D) space to their 2-dimensional (2D) projections on the SPECT detector. Computer-simulation studies and experimental evaluations were performed as a function of number of line sources, Radon transform accuracy, finite line-source width, intrinsic camera resolution, Poisson noise and acquisition geometry. In computer-simulation studies, when there was no error in determining angles (α) and offsets (ρ) of the measured projections, the six alignment parameters (3 translational and 3 rotational) were estimated perfectly using three line sources. When angles (α) and offsets (ρ) were provided by Radon transform, the estimation accuracy was reduced. The estimation error was associated with rounding errors of Radon transform, finite line-source width, Poisson noise, number of line sources, intrinsic camera resolution and detector acquisition geometry. The estimation accuracy was significantly improved by using 4 line sources rather than 3 and also by using thinner line-source projections (obtained by better intrinsic detector resolution). With 5 line sources, median errors were 0.2 mm for the detector translations, 0.7 mm for the detector radius of rotation, and less than 0.5° for detector rotation, tilt and twist. In experimental evaluations, average errors relative to a different, independent registration technique were about 1.8 mm for detector translations, 1.1 mm for the detector radius of rotation (ROR), 0.5° and 0.4° for detector rotation and tilt, respectively, and 1.2° for detector twist.

Simulation studies were performed to investigate the improvement of imaging sensitivity and accuracy of hot sphere localization for breast imaging of patients in prone position. A 3D XCAT phantom was simulated in the prone position with nine hot spheres of 10 mm diameter added in the left breast. A no-treatment-table case and two commercial prone breast boards, 7 and 24 cm thick, were simulated. Different pinhole focal lengths were assessed for root-mean-square-error (RMSE). The pinhole focal lengths resulting in the lowest RMSE values were 12 cm, 18 cm and 21 cm for no table, thin board, and thick board, respectively. In both no table and thin board cases, all 9 hot spheres were easily visualized above background with 4-minute scans utilizing the 49-pinhole SPECT system while seven of nine hot spheres were visible with the thick board. In comparison with parallel-hole system, our 49-pinhole system shows reduction in noise and bias under these simulation cases. These results correspond to smaller radii of rotation for no-table case and thinner prone board. Similarly, localization accuracy with the 49-pinhole system was significantly better than with the parallel-hole system for both the thin and thick prone boards. Median localization errors for the 49-pinhole system with the thin board were less than 3 mm for 5 of 9 hot spheres, and less than 6 mm for the other 4 hot spheres. Median localization errors of 49-pinhole system with the thick board were less than 4 mm for 5 of 9 hot spheres, and less than 8 mm for the other 4 hot spheres.

Besides prone breast imaging, respiratory-gated region-of-interest (ROI) imaging of lung tumor was also investigated. A simulation study was conducted on the potential of multi-pinhole, region-of-interest (ROI) SPECT to alleviate noise effects associated with respiratory-gated SPECT imaging of the thorax. Two 4D XCAT digital phantoms were constructed, with either a 10 mm or 20 mm diameter tumor added in the right lung. The maximum diaphragm motion was 2 cm (for 10 mm tumor) or 4 cm (for 20 mm tumor) in superior-inferior direction and 1.2 cm in anterior-posterior direction. Projections were simulated with a 4-minute acquisition time (40 seconds per each of 6 gates) using either the ROI SPECT system (49-pinhole) or reference single and dual conventional broad cross-section, parallel-hole collimated SPECT. The SPECT images were reconstructed using OSEM with up to 6 iterations. Images were evaluated as a function of gate by profiles, noise versus bias curves, and a numerical observer performing a forced-choice localization task. Even for the 20 mm tumor, the 49-pinhole imaging ROI was found sufficient to encompass fully usual clinical ranges of diaphragm motion. Averaged over the 6 gates, noise at iteration 6 of 49-pinhole ROI imaging (10.9 µCi/ml) was approximately comparable to noise at iteration 2 of the two dual and single parallel-hole, broad cross-section systems (12.4 µCi/ml and 13.8 µCi/ml, respectively). Corresponding biases were much lower for the 49-pinhole ROI system (3.8 µCi/ml), versus 6.2 µCi/ml and 6.5 µCi/ml for the dual and single parallel-hole systems, respectively. Median localization errors averaged over 6 gates, for the 10 mm and 20 mm tumors respectively, were 1.6 mm and 0.5 mm using the ROI imaging system and 6.6 mm and 2.3 mm using the dual parallel-hole, broad cross-section system. The results demonstrate substantially improved imaging via ROI methods. One important application may be gated imaging of patients in position for radiation therapy.

A robotic SPECT imaging system was constructed utilizing a gamma camera detector (Digirad 2020tc) and a robot (KUKA KR150-L110 robot). An imaging study was performed with a phantom (PET CT PhantomTM), which includes 5 spheres of 10, 13, 17, 22 and 28 mm in diameter. The phantom was placed on a flat-top couch. SPECT projections were acquired with a parallel-hole collimator and a single-pinhole collimator both without background in the phantom, and with background at 1/10th the sphere activity concentration. The imaging trajectories of parallel-hole and pinhole collimated detectors spanned 180 degrees and 228 degrees respectively. The pinhole detector viewed a 14.7 cm-diameter common volume which encompassed the 28 mm and 22 mm spheres. The common volume for parallel-hole was a 20.8-cm-diameter cylinder which encompassed all five spheres in the phantom. The maneuverability of the robotic system was tested by navigating the detector to trace the flat-top table while avoiding collision with the table and maintaining the closest possible proximity to the common volume. For image reconstruction, detector trajectories were described by radius-of-rotation and detector rotation angle θ. These reconstruction parameters were obtained from the robot base and tool coordinates. The robotic SPECT system was able to maneuver the parallel-hole and pinhole collimated SPECT detectors in close proximity to the phantom, minimizing impact of the flat-top couch on detector to center-of-rotation (COR) distance. In no background case, all five spheres were visible in the reconstructed parallel-hole and pinhole images. In with background case, three spheres of 17, 22 and 28 mm diameter were readily observed with the parallel-hole imaging, and the targeted spheres (22 and 28 mm diameter) were readily observed in the pinhole ROI imaging.

In conclusion, the proposed on-board robotic SPECT can be aligned to LINAC/CBCT with a single pinhole projection of the line-source phantom. Alignment parameters can be estimated using one pinhole projection of line sources. This alignment method may be important for multi-pinhole SPECT, where relative pinhole alignment may vary during rotation. For single pinhole and multi-pinhole SPECT imaging onboard radiation therapy machines, the method could provide alignment of SPECT coordinates with those of CBCT and the LINAC. In simulation studies of prone breast imaging and respiratory-gated lung imaging, the 49-pinhole detector showed better tumor contrast recovery and localization in a 4-minute scan compared to parallel-hole detector. On-board SPECT could be achieved by a robot maneuvering a SPECT detector about patients in position for radiation therapy on a flat-top couch. The robot inherent coordinate frames could be an effective means to estimate detector pose for use in SPECT image reconstruction.