A Stochastic Model for Electron Multiplication Charge-Coupled Devices - From Theory to Practice

Electron multiplication charge-coupled devices (EMCCD) are widely used for photon counting experiments and measurements of low intensity light sources, and are extensively employed in biological fluorescence imaging applications. These devices have a complex statistical behaviour that is often not fully considered in the analysis of EMCCD data. Robust and optimal analysis of EMCCD images requires an understanding of their noise properties, in particular to exploit fully the advantages of Bayesian and maximum-likelihood analysis techniques, whose value is increasingly recognised in biological imaging for obtaining robust quantitative measurements from challenging data. To improve our own EMCCD analysis and as an effort to aid that of the wider bioimaging community, we present, explain and discuss a detailed physical model for EMCCD noise properties, giving a likelihood function for image counts in each pixel for a given incident intensity, and we explain how to measure the parameters for this model from various calibration images. © 2013 Hirsch et al.

Statistics of single-electron signals in electron-multiplying charge-coupled devices

Electron-multiplying charge coupled devices promise to revolutionize ultrasensitive optical imaging. The authors present a simple methodology allowing reliable measurement of camera characteristics and statistics of single-electron events, compare the measurements to a simple theoretical model, and report camera performance in a truly photon-counting regime that eliminates the excess noise related to fluctuations of the multiplication gain.

DNA-Mediated Charge Transport Devices for Protein Detection

Detection of biologically relevant targets, including small molecules, proteins, DNA, and RNA, is vital for fundamental research as well as clinical diagnostics. Sensors with biological elements provide a natural foundation for such devices because of the inherent recognition capabilities of biomolecules. Electrochemical DNA platforms are simple, sensitive, and do not require complex target labeling or expensive instrumentation. Sensitivity and specificity are added to DNA electrochemical platforms when the physical properties of DNA are harnessed. The inherent structure of DNA, with its stacked core of aromatic bases, enables DNA to act as a wire via DNA-mediated charge transport (DNA CT). DNA CT is not only robust over long molecular distances of at least 34 nm, but is also especially sensitive to anything that perturbs proper base stacking, including DNA mismatches, lesions, or DNA-binding proteins that distort the π-stack. Electrochemical sensors based on DNA CT have previously been used for single-nucleotide polymorphism detection, hybridization assays, and DNA-binding protein detection. Here, improvements to (i) the structure of DNA monolayers and (ii) the signal amplification with DNA CT platforms for improved sensitivity and detection are described.

First, improvements to the control over DNA monolayer formation are reported through the incorporation of copper-free click chemistry into DNA monolayer assembly. As opposed to conventional film formation involving the self-assembly of thiolated DNA, copper-free click chemistry enables DNA to be tethered to a pre-formed mixed alkylthiol monolayer. The total amount of DNA in the final film is directly related to the amount of azide in the underlying alkylthiol monolayer. DNA monolayers formed with this technique are significantly more homogeneous and lower density, with a larger amount of individual helices exposed to the analyte solution. With these improved monolayers, significantly more sensitive detection of the transcription factor TATA binding protein (TBP) is achieved.

Using low-density DNA monolayers, two-electrode DNA arrays were designed and fabricated to enable the placement of multiple DNA sequences onto a single underlying electrode. To pattern DNA onto the primary electrode surface of these arrays, a copper precatalyst for click chemistry was electrochemically activated at the secondary electrode. The location of the secondary electrode relative to the primary electrode enabled the patterning of up to four sequences of DNA onto a single electrode surface. As opposed to conventional electrochemical readout from the primary, DNA-modified electrode, a secondary microelectrode, coupled with electrocatalytic signal amplification, enables more sensitive detection with spatial resolution on the DNA array electrode surface. Using this two-electrode platform, arrays have been formed that facilitate differentiation between well-matched and mismatched sequences, detection of transcription factors, and sequence-selective DNA hybridization, all with the incorporation of internal controls.

For effective clinical detection, the two working electrode platform was multiplexed to contain two complementary arrays, each with fifteen electrodes. This platform, coupled with low density DNA monolayers and electrocatalysis with readout from a secondary electrode, enabled even more sensitive detection from especially small volumes (4 μL per well). This multiplexed platform has enabled the simultaneous detection of two transcription factors, TBP and CopG, with surface dissociation constants comparable to their solution dissociation constants.

With the sensitivity and selectivity obtained from the multiplexed, two working electrode array, an electrochemical signal-on assay for activity of the human methyltransferase DNMT1 was incorporated. DNMT1 is the most abundant human methyltransferase, and its aberrant methylation has been linked to the development of cancer. However, current methods to monitor methyltransferase activity are either ineffective with crude samples or are impractical to develop for clinical applications due to a reliance on radioactivity. Electrochemical detection of methyltransferase activity, in contrast, circumvents these issues. The signal-on detection assay translates methylation events into electrochemical signals via a methylation-specific restriction enzyme. Using the two working electrode platform combined with this assay, DNMT1 activity from tumor and healthy adjacent tissue lysate were evaluated. Our electrochemical measurements revealed significant differences in methyltransferase activity between tumor tissue and healthy adjacent tissue.

As differential activity was observed between colorectal tumor tissue and healthy adjacent tissue, ten tumor sets were subsequently analyzed for DNMT1 activity both electrochemically and by tritium incorporation. These results were compared to expression levels of DNMT1, measured by qPCR, and total DNMT1 protein content, measured by Western blot. The only trend detected was that hyperactivity was observed in the tumor samples as compared to the healthy adjacent tissue when measured electrochemically. These advances in DNA CT-based platforms have propelled this class of sensors from the purely academic realm into the realm of clinically relevant detection.

Analytical Assessment of a Home Made Capillary Electrophoresis Equipment with Linear Charge Coupled Device for Visible Light Absorption Detection in the Determination of Food Dyes

The performance of modular home made capillary electrophoresis equipment with spectrophotometric detection, at a visible region by means of a miniaturized linear charge coupled device, was evaluated for the determination of four food dyes. This system presents a simple but efficient home made cell detection scheme. A computer program that converts the spectral data after each run into the electropherograms was developed to evaluate the analytical parameters. The dyes selected for analytical evaluation of the system were Brilliant Blue FCF, Fast Green FCF, Sunset Yellow FCF, and Amaranth. Separation was carried out in a 29cm length and 75 mu m I.D fused silica capillary, using 10mmolL-1 borate buffer at pH 9, with separation voltage of 7.5kV. The detection limits for the dyes were between 0.3 and 1.5mgL-1 and the method presented adequate linearity over the ranges studied, with correlation coefficients greater than 0.99. The method was applied for determination and quantification of these dyes in fruit juices and candies.

Optical sensor interrogation with a blazed fiber Bragg grating and a charge-coupled device linear array

We present what is to our knowledge the first comprehensive investigation of the use of blazed fiber Bragg gratings (BFBGs) to interrogate wavelength division multiplexed (WDM) in-fiber optical sensor arrays. We show that the light outcoupled from the core of these BFBGs is radiated with sufficient optical power that it may be detected with a low-cost charge-coupled device (CCD) array. We present thorough system performance analysis that shows sufficient spectral-spatial resolution to decode sensors with a WDM separation of 75 ρm, signal-to-noise ratio greater than 45-dB bandwidth of 70 nm, and drift of only 0.1 ρm. We show the system to be polarization-state insensitive, making the BFBG-CCD spectral analysis technique a practical, extremely low-cost, alternative to traditional tunable filter approaches.

Fluorescence-enhanced optical imaging on three-dimensional phantoms using a hand-held probe based frequency-domain intensified charge coupled device (ICCD) optical imager

Fluorescence-enhanced optical imaging is an emerging non-invasive and non-ionizing modality towards breast cancer diagnosis. Various optical imaging systems are currently available, although most of them are limited by bulky instrumentation, or their inability to flexibly image different tissue volumes and shapes. Hand-held based optical imaging systems are a recent development for its improved portability, but are currently limited only to surface mapping. Herein, a novel optical imager, consisting primarily of a hand-held probe and a gain-modulated intensified charge coupled device (ICCD) detector, is developed towards both surface and tomographic breast imaging. The unique features of this hand-held probe based optical imager are its ability to; (i) image large tissue areas (5×10 sq. cm) in a single scan, (ii) reduce overall imaging time using a unique measurement geometry, and (iii) perform tomographic imaging for tumor three-dimensional (3-D) localization. Frequency-domain based experimental phantom studies have been performed on slab geometries (650 ml) under different target depths (1-2.5 cm), target volumes (0.45, 0.23 and 0.10 cc), fluorescence absorption contrast ratios (1:0, 1000:1 to 5:1), and number of targets (up to 3), using Indocyanine Green (ICG) as fluorescence contrast agents. An approximate extended Kalman filter based inverse algorithm has been adapted towards 3-D tomographic reconstructions. Single fluorescence target(s) was reconstructed when located: (i) up to 2.5 cm deep (at 1:0 contrast ratio) and 1.5 cm deep (up to 10:1 contrast ratio) for 0.45 cc-target; and (ii) 1.5 cm deep for target as small as 0.10 cc at 1:0 contrast ratio. In the case of multiple targets, two targets as close as 0.7 cm were tomographically resolved when located 1.5 cm deep. It was observed that performing multi-projection (here dual) based tomographic imaging using a priori target information from surface images, improved the target depth recovery over using single projection based imaging. From a total of 98 experimental phantom studies, the sensitivity and specificity of the imager was estimated as 81-86% and 43-50%, respectively. With 3-D tomographic imaging successfully demonstrated for the first time using a hand-held based optical imager, the clinical translation of this technology is promising upon further experimental validation from in-vitro and in-vivo studies.

Photometric and fundamental plane studies of galaxies

Determining the morphological parameters that describe galaxies has always been a challenging task. The studies on the correlations between different photometric as well as spectroscopic parameters of the galaxies help in understanding their structure, properties of the stars and gas which constitute the galaxy, the various physical and chemical processes which determine the properties, and galaxy formation and evolution. In the last few decades, the advent of Charge Coupled Devices (CCDs) and near infrared arrays ha\·e provided quick and reliable digitized data acquisition, in the optical and near infrared bands. This has provided an avalanche of data, which can be processed using sophisticated image analysis techniques to obtain information about the morphology of galaxies. The photometric analysis performed in this thesis involve the extraction of structural parameters of early type gala.xies imaged in the near infrared K (2.2ttm) band, obtaining correlations between these, parameters and using them to constrain the large scale properties of galaxi,~s.

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.

Digital radiographic evaluation of optical density of porcelains used for fabrication of inlay/onlay prosthesis

The purpose of this article was to compare the mean value of optical density of four porcelains commonly used for fabrication of inlay/ onlay prostheses using direct digital radiograph. The sample consisted of 20 2-mm thick porcelain specimens (measured by digital pachymeter): Empress (Ivoclair), Simbios (Degussa), Vita Omega 900 and Vitadur Alpha (Vita Zahnfabrik). The values of optical density of the specimens were expressed in millimeters aluminum equivalent (mm eq Al). The samples were X-rayed using two charge coupled devices (CCD) - RVG (Trophy) - Visualix (Gendex) and a phosphor plate system - Digora (Soredex). The optical density reading was performed with Image Tool 1.28 in a total of 110 measurements. Statistical analysis showed that there were statistically significant differences in all materials studied (p < 0.05) regardless of the radiographic system used. The highest optical density value was found for Omega 900 (1.8988 mmeqAl - Visualix - Gendex) and the lowest for Vitadur Alpha (0.8647 - Visualix - Gendex). Thus, the material presenting the highest degree of optical density was Omega 900, Empress and Simbios presented intermediate optical density values, Vitadur Alpha presented the lowest value, and the optical density of porcelains was not influenced by the digital radiography systems.

Análise da reconstrução 3D a partir de um par estereoscópico HR-CCD/CBERS-2 usando dois modelos matemáticos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Mammography equipment

Mammography is one of the most technically demanding examinations in radiology, and it requires X-ray technology designed specifi cally for the task. The pathology to be imaged ranges from small (20–100 μm) high density microcalcifications to ill-defi ned low contrast masses. These must be imaged against a background of mixed densities. This makes demonstrating pathology challenging. Because of its use in asymptomatic screening, mammography must also employ as low a radiation dose as possible.