Subcellular imaging of intramitochondrial Ca2+ with recombinant targeted aequorin: significance for the regulation of pyruvate dehydrogenase activity.

Specific targeting of the recombinant, Ca2+ -sensitive photoprotein, aequorin to intracellular organelles has provided new insights into the mechanisms of intracellular Ca2+ homeostasis. When applied to small mammalian cells, a major limitation of this technique has been the need to average the signal over a large number of cells. This prevents the identification of inter- or intracellular heterogeneities. Here we describe the imaging in single mammalian cells (CHO.T) of [Ca2+] with recombinant chimeric aequorin targeted to mitochondria. This was achieved by optimizing expression of the protein through intranuclear injection of cDNA and through the use of a charge-coupled device camera fitted with a dual microchannel plate intensifier. This approach allows accurate quantitation of the kinetics and extent of the large changes in mitochondrial matrix [Ca2+] ([Ca2+](m)) that follow receptor stimulation and reveal different behaviors of mitochondrial populations within individual cells. The technique is compared with measurements of [Ca2+](m) using the fluorescent indicator, rhod2. Comparison of [Ca2+](m) with the activity of the Ca2+ -sensitive matrix enzyme, pyruvate dehydrogenase (PDH), reveals that this enzyme is a target of the matrix [Ca2+] changes. Peak [Ca2+](m) values following receptor stimulation are in excess of those necessary for full activation of PDH in situ, but may be necessary for the activation of other mitochondrial dehydrogenases. Finally, the data suggest that the complex regulation of PDH activity by a phosphorylation-dephosphorylation cycle may provide a means by which changes in the frequency of cytosolic (and hence mitochondrial) [Ca2+] oscillations can be decoded by mitochondria.

DNA analysis and diagnostics on oligonucleotide microchips.

We present a further development in the technology of sequencing by hybridization to oligonucleotide microchips (SHOM) and its application to diagnostics for genetic diseases. A robot has been constructed to manufacture sequencing "microchips." The microchip is an array of oligonucleotides immobilized into gel elements fixed on a glass plate. Hybridization of the microchip with fluorescently labeled DNA was monitored in real time simultaneously for all microchip elements with a two-wavelength fluorescent microscope equipped with a charge-coupled device camera. SHOM has been used to detect beta-thalassemia mutations in patients by hybridizing PCR-amplified DNA with the microchips. A contiguous stacking hybridization technique has been applied for the detection of mutations; it can simplify medical diagnostics and enhance its reliability. The use of multicolor monitoring of contiguous stacking hybridization is suggested for large-scale diagnostics and gene polymorphism studies. Other applications of the SHOM technology are discussed.

Transposable elements are stable structural components of Drosophila melanogaster heterochromatin.

We determined the distribution of 11 different transposable elements on Drosophila melanogaster mitotic chromosomes by using high-resolution fluorescent in situ hybridization (FISH) coupled with charge-coupled device camera analysis. Nine of these transposable elements (copia, gypsy, mdg-1, blood, Doc, I, F, G, and Bari-1) are preferentially clustered into one or more discrete heterochromatic regions in chromosomes of the Oregon-R laboratory stock. Moreover, FISH analysis of geographically distant strains revealed that the locations of these heterochromatic transposable element clusters are highly conserved. The P and hobo elements, which are likely to have invaded the D. melanogaster genome at the beginning of this century, are absent from Oregon-R heterochromatin but clearly exhibit heterochromatic clusters in certain natural populations. Together these data indicate that transposable elements are major structural components of Drosophila heterochromatin, and they change the current views on the role of transposable elements in host genome evolution.

In situ imaging and height reconstruction of phase separation processes in polymer blends during spin coating

Spin coating polymer blend thin films provides a method to produce multiphase functional layers of high uniformity covering large surface areas. Applications for such layers include photovoltaics and light-emitting diodes where performance relies upon the nanoscale phase separation morphology of the spun film. Furthermore, at micrometer scales, phase separation provides a route to produce self-organized structures for templating applications. Understanding the factors that determine the final phase-separated morphology in these systems is consequently an important goal. However, it has to date proved problematic to fully test theoretical models for phase separation during spin coating, due to the high spin speeds, which has limited the spatial resolution of experimental data obtained during the coating process. Without this fundamental understanding, production of optimized micro- and nanoscale structures is hampered. Here, we have employed synchronized stroboscopic illumination together with the high light gathering sensitivity of an electron-multiplying charge-coupled device camera to optically observe structure evolution in such blends during spin coating. Furthermore the use of monochromatic illumination has allowed interference reconstruction of three-dimensional topographies of the spin-coated film as it dries and phase separates with nanometer precision. We have used this new method to directly observe the phase separation process during spinning for a polymer blend (PS-PI) for the first time, providing new insights into the spin-coating process and opening up a route to understand and control phase separation structures. © 2011 American Chemical Society.

VAMP - a vision based sensor network for health care hygiene

Adequate hand-washing has been shown to be a critical activity in preventing the transmission of infections such as MRSA in health-care environments. Hand-washing guidelines published by various health-care related institutions recommend a technique incorporating six hand-washing poses that ensure all areas of the hands are thoroughly cleaned. In this paper, an embedded wireless vision system (VAMP) capable of accurately monitoring hand-washing quality is presented. The VAMP system hardware consists of a low resolution CMOS image sensor and FPGA processor which are integrated with a microcontroller and ZigBee standard wireless transceiver to create a wireless sensor network (WSN) based vision system that can be retargeted at a variety of health care applications. The device captures and processes images locally in real-time, determines if hand-washing procedures have been correctly undertaken and then passes the resulting high-level data over a low-bandwidth wireless link. The paper outlines the hardware and software mechanisms of the VAMP system and illustrates that it offers an easy to integrate sensor solution to adequately monitor and improve hand hygiene quality. Future work to develop a miniaturized, low cost system capable of being integrated into everyday products is also discussed.

Novel smart modules for imaging, communications, and displays

This dissertation proposes and demonstrates novel smart modules to solve challenging problems in the areas of imaging, communications, and displays. The smartness of the modules is due to their ability to be able to adapt to changes in operating environment and application using programmable devices, specifically, electronically variable focus lenses (ECVFLs) and digital micromirror devices (DMD). The proposed modules include imagers for laser characterization and general purpose imaging which smartly adapt to changes in irradiance, optical wireless communication systems which can adapt to the number of users and to changes in link length, and a smart laser projection display that smartly adjust the pixel size to achieve a high resolution projected image at each screen distance. The first part of the dissertation starts with the proposal of using an ECVFL to create a novel multimode laser beam characterizer for coherent light. This laser beam characterizer uses the ECVFL and a DMD so that no mechanical motion of optical components along the optical axis is required. This reduces the mechanical motion overhead that traditional laser beam characterizers have, making this laser beam characterizer more accurate and reliable. The smart laser beam characterizer is able to account for irradiance fluctuations in the source. Using image processing, the important parameters that describe multimode laser beam propagation have been successfully extracted for a multi-mode laser test source. Specifically, the laser beam analysis parameters measured are the M2 parameter, w0 the minimum beam waist, and zR the Rayleigh range. Next a general purpose incoherent light imager that has a high dynamic range (>100 dB) and automatically adjusts for variations in irradiance in the scene is proposed. Then a data efficient image sensor is demonstrated. The idea of this smart image sensor is to reduce the bandwidth needed for transmitting data from the sensor by only sending the information which is required for the specific application while discarding the unnecessary data. In this case, the imager demonstrated sends only information regarding the boundaries of objects in the image so that after transmission to a remote image viewing location, these boundaries can be used to map out objects in the original image. The second part of the dissertation proposes and demonstrates smart optical communications systems using ECVFLs. This starts with the proposal and demonstration of a zero propagation loss optical wireless link using visible light with experiments covering a 1 to 4 m range. By adjusting the focal length of the ECVFLs for this directed line-of-sight link (LOS) the laser beam propagation parameters are adjusted such that the maximum amount of transmitted optical power is captured by the receiver for each link length. This power budget saving enables a longer achievable link range, a better SNR/BER, or higher power efficiency since more received power means the transmitted power can be reduced. Afterwards, a smart dual mode optical wireless link is proposed and demonstrated using a laser and LED coupled to the ECVFL to provide for the first time features of high bandwidths and wide beam coverage. This optical wireless link combines the capabilities of smart directed LOS link from the previous section with a diffuse optical wireless link, thus achieving high data rates and robustness to blocking. The proposed smart system can switch from LOS mode to Diffuse mode when blocking occurs or operate in both modes simultaneously to accommodate multiple users and operate a high speed link if one of the users requires extra bandwidth. The last part of this section presents the design of fibre optic and free-space optical switches which use ECVFLs to deflect the beams to achieve switching operation. These switching modules can be used in the proposed optical wireless indoor network. The final section of the thesis presents a novel smart laser scanning display. The ECVFL is used to create the smallest beam spot size possible for the system designed at the distance of the screen. The smart laser scanning display increases the spatial resoluti on of the display for any given distance. A basic smart display operation has been tested for red light and a 4X improvement in pixel resolution for the image has been demonstrated.

A method for the digital image analysis of ceramic grains based on shape factor segmentation

A very simple and robust method for ceramics grains quantitative image analysis is presented. Based on the use of optimal imaging conditions for reflective light microscopy of bulk samples, a digital image processing routine was developed for shading correction, noise suppressing and contours enhancement. Image analysis was done for grains selected according to their concavities, evaluated by perimeter ratio shape factor, to avoid consider the effects of breakouts and ghost boundaries due to ceramographic preparation limitations. As an example, the method was applied for two ceramics, to compare grain size and morphology distributions. In this case, most of artefacts introduced by ceramographic preparation could be discarded due to the use of perimeter ratio exclusion range.

Fiber Bragg grating sensor interrogation system using a CCD side detection method with Superimposed blazed gratings

We present, for the first time to our knowledge, experimental evidence showing that superimposed blazed fiber Bragg gratings may be fabricated and used to extend the dynamic range of a grating-based spectrometer. Blazed gratings of 4° and 8° were superimposed in germanosilicate fiber by ultraviolet inscription and used in conjunction with a coated charged-coupled device array to interrogate a wavelength-division-multiplexing sensor array. We show that the system can be used to monitor strain and temperature sensors simultaneously with an employable bandwidth which is extendable to 70 nm.

Fiber Bragg grating sensor interrogation system using a CCD side detection method with superimposed blazed gratings

We present, for the first time to our knowledge, experimental evidence showing that superimposed blazed fiber Bragg gratings may be fabricated and used to extend the dynamic range of a grating-based spectrometer. Blazed gratings of 4° and 8° were superimposed in germanosilicate fiber by ultraviolet inscription and used in conjunction with a coated charged-coupled device array to interrogate a wavelength-division-multiplexing sensor array. We show that the system can be used to monitor strain and temperature sensors simultaneously with an employable bandwidth which is extendedable to 70 nm.

Eye-view: A retinal image acquisition system

This paper presents a low cost but high resolution retinal image acquisition system of the human eye. The images acquired by a CMOS image sensor are communicated through the Universal Serial Bus (USB) interface to a personal computer for viewing and further processing. The image acquisition time was estimated to be 2.5 seconds. This system can also be used in telemedicine applications.

Eye-View: A retinal image capture system

This paper presents a low cost but high resolution retinal image acquisition system of the human eye. The images acquired by a CMOS image sensor are communicated through the Universal Serial Bus (USB) interface to a personal computer for viewing and further processing. The image acquisition time was estimated to be 2.5 seconds. This system can also be used in telemedicine applications.

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.

A high-speed image sensing technique with adjustable frame rate based on an ordinary CCD

In the sinusoidal phase modulating interferometer technique, the high-speed CCD is necessary to detect the interference signals. The reason of ordinary CCD's low frame rate was analyzed, and a novel high-speed image sensing technique with adjustable frame rate based on ail ordinary CCD was proposed. And the principle of the image sensor was analyzed. When the maximum frequency and channel bandwidth were constant, a custom high-speed sensor was designed by using the ordinary CCD under the control of the special driving circuit. The frame rate of the ordinary CCD has been enhanced by controlling the number of pixels of every frame; therefore, the ordinary of CCD can be used as the high frame rate image sensor with small amount of pixels. The multi-output high-speed image sensor has the deficiencies of low accuracy, and high cost, while the high-speed image senor with small number of pixels by using this technique can overcome theses faults. The light intensity varying with time was measured by using the image sensor. The frame rate was LIP to 1600 frame per second (f/s), and the size of every frame and the frame rate were adjustable. The correlation coefficient between the measurement result and the standard values were higher than 0.98026, and the relative error was lower than 0.53%. The experimental results show that this sensor is fit to the measurements of sinusoidal phase modulating interferometer technique. (c) 2007 Elsevier GmbH. All rights reserved.

Butt-coupled MOVPE growth for high-performance electro-absorption modulator integrated with a DFB laser

A novel butt-joint coupling scheme is proposed to improve the coupling efficiency for the integration of a GalnAsP MQW distributed feedback (DFB) laser with an MQW electro-absorption modulator (EAM). The proposed method gives more than 90% coupling efficiency, being much higher than the 26% coupling efficiency of the common MQW-MQW coupling technique. The differential quantum efficiency of the MQW-bulk-MQW coupled device is also much higher than that of the MQW-MQW device, 0.106 mW/mA versus 0.02 mW/mA. The EAM-DFB devices fabricated by the proposed method exhibit a very high modulation efficiency (12 dB/V) from 0 to I V. By adopting a high-mesa ridge waveguide and buried polyimide, the capacitance of the modulator is reduced to about 0.28 pF. The experimental results demonstrate that the method can replace the conventional MQW-MQW coupling technique to fabricate high-quality integrated photonic devices. (C) 2007 Elsevier B.V. All rights reserved.

Thomson scattering system at the Tokyo electron beam ion trap

A Thomson scattering system has been installed at the Tokyo electron beam ion trap for probing characteristics of the electron beam. A YVO4 green laser beam was injected antiparallel to the electron beam. The image of the Thomson scattering light from the electron beam has been observed using a charged-coupled device camera. By using a combination of interference filters, the spectral distribution of the Thomson scattering light has been measured. The Doppler shift observed for the scattered light is consistent with the beam energy. The beam radius dependence was investigated as a function of the beam energy, the beam current, and the magnetic field at the trap region. The variation of the measured beam radius against the beam current and the magnetic field were similar to those in Herrmann's prediction. The beam radius as a function of the beam energy was also similar to Herrmann's prediction but seemed to become larger at low energy. (C) 2002 American Institute of Physics.