Biotechnologies for cancer diagnostics : cell sorting, protein analysis and imaging of cellular metabolism

This thesis presents the development of chip-based technology for informative in vitro cancer diagnostics. In the first part of this thesis, I will present my contribution in the development of a technology called “Nucleic Acid Cell Sorting (NACS)”, based on microarrays composed of nucleic acid encoded peptide major histocompatibility complexes (p/MHC), and the experimental and theoretical methods to detect and analyze secreted proteins from single or few cells.

Secondly, a novel portable platform for imaging of cellular metabolism with radio probes is presented. A microfluidic chip, so called “Radiopharmaceutical Imaging Chip” (RIMChip), combined with a beta-particle imaging camera, is developed to visualize the uptake of radio probes in a small number of cells. Due to its sophisticated design, RIMChip allows robust and user-friendly execution of sensitive and quantitative radio assays. The performance of this platform is validated with adherent and suspension cancer cell lines. This platform is then applied to study the metabolic response of cancer cells under the treatment of drugs. Both cases of mouse lymphoma and human glioblastoma cell lines, the metabolic responses to the drug exposures are observed within a short time (~ 1 hour), and are correlated with the arrest of cell-cycle, or with changes in receptor tyrosine kinase signaling.

The last parts of this thesis present summaries of ongoing projects: development of a new agent as an in vivo imaging probe for c-MET, and quantitative monitoring of glycolytic metabolism of primary glioblastoma cells. To develop a new agent for c-MET imaging, the one-bead-one-compound combinatorial library method is used, coupled with iterative screening. The performance of the agent is quantitatively validated with cell-based fluorescent assays. In the case of monitoring the metabolism of primary glioblastoma cell, by RIMChip, cells were sorting according to their expression levels of oncoprotein, or were treated with different kinds of drugs to study the metabolic heterogeneity of cancer cells or metabolic response of glioblastoma cells to drug treatments, respectively.

Heterodimerization of integrin Mac-1 subunits studied by single-molecule imaging

Heterodimerization of integrin Mac-1 (alpha(M) beta(2)) Subunits plays important role on regulating leukocytes adhesion to extracellular matrix or endothelial cells. Here, using total internal reflection microscopy, we investigated the heterodimerization of integrin Mac-1 subunits at the single-molecule level in live cells. Individual alpha(M) subunit fused to the enhanced yellow fluorescent protein (eYFP) was imaged at the basal plasma membrane of live Chinese hamster ovary (CHO) cells. Through analysis of mean square displacement (MSD), diffusion coefficient, the size of restricted domain and fraction of molecules undergoing restricted diffusion, we found that as compared with the diffusion in the absence of beta(2) subunit, the diffusion of single-molecule of alpha(M)-YFP was suppressed significantly in the presence of beta(2) subunit. Thus, based on the oligomerization-induced trapping model, we suggested that in the presence of beta(2) subunit, the am subunit may form heterodimer with it. (C) 2008 Elsevier Inc. All rights reserved.

Seismic reflection experiments imaging the physical nature of crustal structures in southern California

Seismic reflection methods have been extensively used to probe the Earth's crust and suggest the nature of its formative processes. The analysis of multi-offset seismic reflection data extends the technique from a reconnaissance method to a powerful scientific tool that can be applied to test specific hypotheses. The treatment of reflections at multiple offsets becomes tractable if the assumptions of high-frequency rays are valid for the problem being considered. Their validity can be tested by applying the methods of analysis to full wave synthetics.

Three studies illustrate the application of these principles to investigations of the nature of the crust in southern California. A survey shot by the COCORP consortium in 1977 across the San Andreas fault near Parkfield revealed events in the record sections whose arrival time decreased with offset. The reflectors generating these events are imaged using a multi-offset three-dimensional Kirchhoff migration. Migrations of full wave acoustic synthetics having the same limitations in geometric coverage as the field survey demonstrate the utility of this back projection process for imaging. The migrated depth sections show the locations of the major physical boundaries of the San Andreas fault zone. The zone is bounded on the southwest by a near-vertical fault juxtaposing a Tertiary sedimentary section against uplifted crystalline rocks of the fault zone block. On the northeast, the fault zone is bounded by a fault dipping into the San Andreas, which includes slices of serpentinized ultramafics, intersecting it at 3 km depth. These interpretations can be made despite complications introduced by lateral heterogeneities.

In 1985 the Calcrust consortium designed a survey in the eastern Mojave desert to image structures in both the shallow and the deep crust. Preliminary field experiments showed that the major geophysical acquisition problem to be solved was the poor penetration of seismic energy through a low-velocity surface layer. Its effects could be mitigated through special acquisition and processing techniques. Data obtained from industry showed that quality data could be obtained from areas having a deeper, older sedimentary cover, causing a re-definition of the geologic objectives. Long offset stationary arrays were designed to provide reversed, wider angle coverage of the deep crust over parts of the survey. The preliminary field tests and constant monitoring of data quality and parameter adjustment allowed 108 km of excellent crustal data to be obtained.

This dataset, along with two others from the central and western Mojave, was used to constrain rock properties and the physical condition of the crust. The multi-offset analysis proceeded in two steps. First, an increase in reflection peak frequency with offset is indicative of a thinly layered reflector. The thickness and velocity contrast of the layering can be calculated from the spectral dispersion, to discriminate between structures resulting from broad scale or local effects. Second, the amplitude effects at different offsets of P-P scattering from weak elastic heterogeneities indicate whether the signs of the changes in density, rigidity, and Lame's parameter at the reflector agree or are opposed. The effects of reflection generation and propagation in a heterogeneous, anisotropic crust were contained by the design of the experiment and the simplicity of the observed amplitude and frequency trends. Multi-offset spectra and amplitude trend stacks of the three Mojave Desert datasets suggest that the most reflective structures in the middle crust are strong Poisson's ratio (σ) contrasts. Porous zones or the juxtaposition of units of mutually distant origin are indicated. Heterogeneities in σ increase towards the top of a basal crustal zone at ~22 km depth. The transition to the basal zone and to the mantle include increases in σ. The Moho itself includes ~400 m layering having a velocity higher than that of the uppermost mantle. The Moho maintains the same configuration across the Mojave despite 5 km of crustal thinning near the Colorado River. This indicates that Miocene extension there either thinned just the basal zone, or that the basal zone developed regionally after the extensional event.

Bright-field and fluorescence chip-scale microscopy for biological imaging

Optical microscopy is an essential tool in biological science and one of the gold standards for medical examinations. Miniaturization of microscopes can be a crucial stepping stone towards realizing compact, cost-effective and portable platforms for biomedical research and healthcare. This thesis reports on implementations of bright-field and fluorescence chip-scale microscopes for a variety of biological imaging applications. The term “chip-scale microscopy” refers to lensless imaging techniques realized in the form of mass-producible semiconductor devices, which transforms the fundamental design of optical microscopes.

Our strategy for chip-scale microscopy involves utilization of low-cost Complementary metal Oxide Semiconductor (CMOS) image sensors, computational image processing and micro-fabricated structural components. First, the sub-pixel resolving optofluidic microscope (SROFM), will be presented, which combines microfluidics and pixel super-resolution image reconstruction to perform high-throughput imaging of fluidic samples, such as blood cells. We discuss design parameters and construction of the device, as well as the resulting images and the resolution of the device, which was 0.66 µm at the highest acuity. The potential applications of SROFM for clinical diagnosis of malaria in the resource-limited settings is discussed.

Next, the implementations of ePetri, a self-imaging Petri dish platform with microscopy resolution, are presented. Here, we simply place the sample of interest on the surface of the image sensor and capture the direct shadow images under the illumination. By taking advantage of the inherent motion of the microorganisms, we achieve high resolution (~1 µm) imaging and long term culture of motile microorganisms over ultra large field-of-view (5.7 mm × 4.4 mm) in a specialized ePetri platform. We apply the pixel super-resolution reconstruction to a set of low-resolution shadow images of the microorganisms as they move across the sensing area of an image sensor chip and render an improved resolution image. We perform longitudinal study of Euglena gracilis cultured in an ePetri platform and image based analysis on the motion and morphology of the cells. The ePetri device for imaging non-motile cells are also demonstrated, by using the sweeping illumination of a light emitting diode (LED) matrix for pixel super-resolution reconstruction of sub-pixel shifted shadow images. Using this prototype device, we demonstrate the detection of waterborne parasites for the effective diagnosis of enteric parasite infection in resource-limited settings.

Then, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope, which uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is also based on the image reconstruction with sweeping illumination technique, where the sequence of images are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.

Finally, we report on the implementation of fluorescence chip-scale microscope, based on a silo-filter structure fabricated on the pixel array of a CMOS image sensor. The extruded pixel design with metal walls between neighboring pixels successfully guides fluorescence emission through the thick absorptive filter to the photodiode layer of a pixel. Our silo-filter CMOS image sensor prototype achieves 13-µm resolution for fluorescence imaging over a wide field-of-view (4.8 mm × 4.4 mm). Here, we demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.

Near-field x-ray phase contrast imaging and phase retrieval algorithm

Theoretical analyses of x-ray diffraction phase contrast imaging and near field phase retrieval method are presented. A new variant of the near field intensity distribution is derived with the optimal phase imaging distance and spatial frequency of object taken into account. Numerical examples of phase retrieval using simulated data are also given. On the above basis, the influence of detecting distance and polychroism of radiation on the phase contrast image and the retrieved phase distribution are discussed. The present results should be useful in the practical application of in-line phase contrast imaging.

硬X射线相位衬度成像的实验研究

介绍了硬X射线(类同轴)相位衬度成像的工作原理及其实验研究结果。X射线波长为0．08860nm,样品为未经任何处理的飞蛾，记录介质为X射线胶片。胶片经处理以后，用光学显微镜读出，可以看出样品的许多细节,尤其在折射率突变处。而同样条件下基于吸收衬度机制的硬X射线吸收成像，由于是弱吸收样品．没有观察到任何图像。

Imaging and tracking single fluorescent nanobeads in solution

In single-particle tracking (SPT), fluorescence video microscopy is used to record the motion images of single particle or single molecule. Here, by using a total-internal-reflection microscope equipped with an argon ion laser and a charge-coupled device (CCD) camera with high-speed and high-sensitivity, video images of single nanobeads in solutions were obtained. From the trajectories, the diffusion coefficient of individual nanobead was determined by the mean square displacements as a function of time. The sizes of nanobeads were calculated by Stokes-Einstein equation, and the results were compared with the actual values.

Innovations of wide-field optical-sectioning fluorescence microscopy : toward high-speed volumetric bio-imaging with simplicity

Optical microscopy has become an indispensable tool for biological researches since its invention, mostly owing to its sub-cellular spatial resolutions, non-invasiveness, instrumental simplicity, and the intuitive observations it provides. Nonetheless, obtaining reliable, quantitative spatial information from conventional wide-field optical microscopy is not always intuitive as it appears to be. This is because in the acquired images of optical microscopy the information about out-of-focus regions is spatially blurred and mixed with in-focus information. In other words, conventional wide-field optical microscopy transforms the three-dimensional spatial information, or volumetric information about the objects into a two-dimensional form in each acquired image, and therefore distorts the spatial information about the object. Several fluorescence holography-based methods have demonstrated the ability to obtain three-dimensional information about the objects, but these methods generally rely on decomposing stereoscopic visualizations to extract volumetric information and are unable to resolve complex 3-dimensional structures such as a multi-layer sphere.

The concept of optical-sectioning techniques, on the other hand, is to detect only two-dimensional information about an object at each acquisition. Specifically, each image obtained by optical-sectioning techniques contains mainly the information about an optically thin layer inside the object, as if only a thin histological section is being observed at a time. Using such a methodology, obtaining undistorted volumetric information about the object simply requires taking images of the object at sequential depths.

Among existing methods of obtaining volumetric information, the practicability of optical sectioning has made it the most commonly used and most powerful one in biological science. However, when applied to imaging living biological systems, conventional single-point-scanning optical-sectioning techniques often result in certain degrees of photo-damages because of the high focal intensity at the scanning point. In order to overcome such an issue, several wide-field optical-sectioning techniques have been proposed and demonstrated, although not without introducing new limitations and compromises such as low signal-to-background ratios and reduced axial resolutions. As a result, single-point-scanning optical-sectioning techniques remain the most widely used instrumentations for volumetric imaging of living biological systems to date.

In order to develop wide-field optical-sectioning techniques that has equivalent optical performance as single-point-scanning ones, this thesis first introduces the mechanisms and limitations of existing wide-field optical-sectioning techniques, and then brings in our innovations that aim to overcome these limitations. We demonstrate, theoretically and experimentally, that our proposed wide-field optical-sectioning techniques can achieve diffraction-limited optical sectioning, low out-of-focus excitation and high-frame-rate imaging in living biological systems. In addition to such imaging capabilities, our proposed techniques can be instrumentally simple and economic, and are straightforward for implementation on conventional wide-field microscopes. These advantages together show the potential of our innovations to be widely used for high-speed, volumetric fluorescence imaging of living biological systems.

The Equivalency Principle for Discounting the Value of Natural Assets: An Application to an Investment Project in the Basque Coast

25 p.

超连续谱干涉方法测量古依相移

利用两束超连续光干涉得到的信号,测量了透镜焦点附近飞秒激光脉冲的古依(Gouy)相移。根据得到的光谱干涉信号,利用傅里叶变换得到相对相位值。激光光束在聚焦透镜后的束腰半径可以由成像方法测得。根据测量得到的激光光束束腰半径,用非线性拟合的方法得到了古依相移曲线,拟合曲线与实验结果符合得非常好。给出了古依相位在焦点前后1 mm区域内的移动量。

Phase imaging with partially coherent x rays

X-ray phase imaging with illumination by a partially coherent source with a setup similar to in-line holography is considered. Using the optical transform function, we consider the effects of partial coherence on this x-ray phase imaging for a weak phase object. The optimal contrast and the resolution of phase imaging are analyzed. As the coherence decreases, the imaging contrast and the optimal contrast frequency decrease, and the resolution degrades. It is shown that this contrast-enhanced phase-imaging method can be regarded as a linear bandpass filter and that the bandwidth and the image contrast are changeable. The frequency property of the imaging system can be improved if an incoherent x-ray source with the proper shape is used. (C) 1999 Optical Society of America.

Instability of solution of phase retrieval in direct diffraction phase-contrast imaging with partially coherent X-ray source

The theoretical model of direct diffraction phase-contrast imaging with partially coherent x-ray source is expressed by an operator of multiple integral. It is presented that the integral operator is linear. The problem of its phase retrieval is described by solving an operator equation of multiple integral. It is demonstrated that the solution of the phase retrieval is unstable. The numerical simulation is performed and the result validates that the solution of the phase retrieval is unstable.

Resolution and noise in ghost imaging with classical thermal light

The resolution and classical noise in ghost imaging with a classical thermal light are investigated theoretically. For ghost imaging with a Gaussian Schell model source, the dependences of the resolution and noise on the spatial coherence of the source and the aperture in the imaging system are discussed and demonstrated by using numerical simulations. The results show that an incoherent source and a large aperture will lead to a good image quality and small noise.

Cross spectral purity and its influence on ghost imaging experiments

Pseudo-thermal light has been widely used in ghost imaging experiments. In order to understand the differences between the pseudo-thermal source and thermal source, we propose a method to investigate whether a light source has cross spectral purity (CSP), and experimentally measure the cross spectral properties of the pseudo-thermal light source in near-field and far-field zones. Moreover we present a theoretical analysis of the cross spectral influence on ghost imaging. (c) 2006 Elsevier B.V. All rights reserved.

Transmission area and correlated imaging

The relationship between transmission area of an object imaged and the visibility of correlated imaging is investigated in a lensless system. We show that they are not in simple inverse proportion, as usually depicted. The changes of the visibility will be quite different when the transmission area is varied by different manners, which may motivate people to seek a new understanding about the influence factors of the visibility. (C) 2007 Optical Society of America