John L. A. Fordham

Screening Autoantibody Profiles in Systemic Rheumatic Disease with a Diagnostic Protein Microarray That Uses a Filtration-Assisted Nanodot Array Luminometric Immunoassay (NALIA)

BACKGROUND We developed a cost-efficient modular system for multiplex analysis of the multiple autoantibodies that characterize systemic rheumatoid diseases. METHODS The nanodot array luminometric immunoassay (NALIA) system consists of conventional 96-well membrane-bottomed plates in which antigens or antibodies are adsorbed onto the underside of the membrane. Current arrays use a 5 x 5 format (25 dots/well), which allows 10 analytes to be measured in duplicate: double-stranded DNA (dsDNA), centromere protein B (CENP-B), PCNA, Sm, Sm ribonucleoprotein (Sm-RNP), U1-snRNP, Scl70, SSA/Ro, SSB/La, Jo-1, and controls. The test fluid, control sera, and subsequent reagents are drawn through the membrane. The captured analytes are quantified by monitoring chemiluminescence with a charge-coupled device (CCD) and analyzed with commercial array software. RESULTS The assay can detect <20 x 10(3) IU/L of anti-dsDNA. The interwell CV was 10%-14%. There was an 83% concordance (kappa = 0.56) between the NALIA results obtained for anti-dsDNA assayed by beta-testing in a routine immunology diagnostic laboratory and the results obtained with a conventional ELISA reagent set. The concordance values for Ro, La, Sm, and RNP were 98% (kappa, 0.92), 93% (kappa, 0.41), 97% (kappa, 0.62), and 97% (kappa, 0.73), respectively. CONCLUSION The NALIA approach promises to provide a highly economical platform for a wide range of applications that require assays of multiple analytes. The degree of concordance of our results with a conventional reagent set was no less than that occurring between different commercial products. A sample of serum from a finger stick provides a volume sufficient to perform the array assay.

The effect of event shape on controiding in photon counting detectors

Abstract High resolution, CCD readout, photon counting detectors employ simple centroiding algorithms for defining the spatial position of each detected event. The accuracy of centroiding is very dependent upon a number of parameters including the profile, energy and width of the intensified event. In this paper, we provide an analysis of how the characteristics of an intensified event change as the input count rate increases and the consequent effect on centroiding. The changes in these parameters are applied in particular to the MIC photon counting detector developed at UCL for ground and space based astronomical applications. This detector has a maximum format of 3072 × 2304 pixels permitting its use in the highest resolution applications. Individual events, at light level from 5 to 1000k events/s over the detector area, were analysed. It was found that both the asymmetry and width of event profiles were strongly dependent upon the energy of the intensified event. The variation in profile then affected the centroiding accuracy leading to loss of resolution. These inaccuracies have been quantified for two different 3 CCD pixel centroiding algorithms and one 2 pixel algorithm. The results show that a maximum error of less than 0.05 CCD pixel occurs with the 3 pixel algorithms and 0.1 CCD pixel for the 2 pixel algorithm. An improvement is proposed by utilising straight pore MCPs in the intensifier and a 70 μm air gap in front of the CCD.

A low budget luminometer for sensitive chemiluminescent immunoassays

We have designed a simple luminometer based on a reasonably priced Peltier-cooled charge-coupled device (CCD) camera, housed in a light-tight box, with straightforward lens imaging and a simple platform for a microtitre or other assay format. The quantitative readout of the CCD image is recorded on a PC using customised software. The instrument can be assembled in a standard university workshop for under pound3000, compared with the cheapest commercial instruments retailing at pound10,000 and above. Consistent with the general view on chemiluminescent assays, the sensitivity is 10-100 times greater than that obtained with parallel ELISAs using a chromogenic substrate. A unique feature of the CCD format is that it enables assays to be carried out on arrays of minidots and even nanodots of antigen on the floor of each microtitre well. This permits direct comparison and standardisation of reactivity of a single sample against several antigens and economy in the use of reagents, test sample and technician time; finger-prick samples of blood can be analysed. The instrument should have widespread applicability in developing countries and, indeed, in any laboratories with hard-pressed budgets.

Phase-resolved Spectroscopic Imaging of the Crab Pulsar

High time resolution spectroscopic imaging of the Crab pulsar across the optical wave band has been undertaken utilizing the 4 m Mayall telescope at Kitt Peak National Observatory. The 33.51 ms period of the pulsar was split into 723.7 time slices of 46.3 μs and a spectrum within each slice acquired. These data have allowed analysis of both the light curve as a function of wavelength and the spectrum as a function of phase position. Wavelength-dependent changes in phase position for both emission peaks have been found, and these appear to be uncorrelated. Related to these changes are phase position-dependent variations in the spectral index, and these have been measured, the shapes being in agreement with previously published data. Also related are energy-dependent changes in the FWHMs. In addition, we find, in support of previously published data, that the fluence of Peak 2 relative to Peak 1 decreases with increasing wavelength. These results then challenge the validity of currently existing models for pulsar emission.

MIC photon counting detector

The MIC photon counting detector, a very high resolution, large format system that has been developed for astronomical applications and has been proven on the major UK associated telescopes, is described. Additionally, though, this detector does have a number of applications in other fields such as bio-medical and x-ray imaging. The detector itself consists of a specially designed 40 mm diameter micro-channel plate intensifier fiber optically coupled to a CCD read-out system. Data is then centroided to 1/8th of a CCD pixel in both X and Y to provide high resolution. Accumulated data is stored in a micro-processor system with on-line display and reduction facilities. The maximum format available with the detector is 3072 X 2304 pixels, where each pixel is 10.6 micrometers square. The resolution is 27 micrometers FWHM when averaged over the field. Dependent upon the application, a dynamic range as high as 5 X 106 is achievable with this detector. The time resolution of the detector is in the range 1 ms to 12 ms. A very large format version of this detector is being designed that utilizes a 75 mm intensifier and has a maximum format of 6144 X 4608 pixels. It is expected that this detector will have the same performance figures as the 40 mm system.

DQE enhancement of MCP intensifiers for astronomy results of the MIC II program

The microchannel plate intensified CCD (MIC) photon counting detector system was developed to replace a common user photon counting detector, the image photon counting system (IPCS), at the Anglo Australian Telescope and at the William Herschel Telescope and the Isaac Newton Telescope at La Palma Observatory. The IPCS incorporated magnetically-focused four-stage cascade image intensifiers. This paper discusses technological aspects of the design and optimization of very high gain microchannel plate image intensifiers for such photon counting systems and particularly the optimization of device detective quantum efficiency.

MCP image intensifier with improved DQE

The Microchannel-plate Intensified CCD (MIC) photon-counting detector system has been developed as a future replacement for a common user photon counting detector, the IPCS (Image Photon Counting System) on both the Isaac Newton and William Herschel telescopes at the La Palma Observatory and at the Anglo-Australian Observatory. These detectors previously incorporated EMI 4-stage cascade image intensifiers. This paper addresses the technological aspects of the design and optimization of very high gain MCP image intensifiers for such photon counting systems, and particularly the optimization of device detective quantum efficiency.

Evaluation of Photon-event-counting Intensifiers

Publisher Summary This chapter discusses the evaluation of photon-event-counting intensifiers. It is proposed to consider some of the measurements that have been carried out to evaluate the performance of the detector for photon-event counting. Measurements carried out on the prototype 40 mm microchannel plate (MCP) image intensifiers that have been produced as part of a programme to develop an improved front-end detector for photon-event-counting systems indicate that the tubes have several desirable characteristics for use in this application. Device operating parameters such as gain, resolution, pulse height distribution, noise, and detective quantum efficiency can be measured. Light entering the device may suffer several reflections in the intensifier input faceplate or inside the vacuum envelope. Measurements have been made of a number of critical parameters that are of particular importance for photon-event-counting systems and that are often not quoted by commercial manufacturers. MCP counting efficiency can be improved by modifications to the manufacturing procedures.

Performance of a large-area microchannel plate photon-counting intensifier

A 75 mm diameter microchannel plate (MCP) intensifier has been developed for astronomical applications. The intensifier incorporates a semi-transparent photocathode, three MCPs in a Z- stack configuration, and a P20 phosphor screen in a dual proximity focused arrangement. The input MCP is a thin 40:1 channel plate which is conditioned to run at low gain and hence act as an ion barrier for the succeeding 80:1 chevron pair. The intensifier has been incorporated into a CCD readout system and has undergone extensive laboratory testing. The preconditioning of these 75 mm diameter channel plates required a large area, highly uniform electron scrub beam, this has led to the development of a novel electron gun. The design of the 75 mm intensifier and the novel electron gun are described. Results from the laboratory evaluation of the intensifier are presented. Flat field illumination showed the existence of self- exciting channels in a hexagonal pattern. Finally, a future UV or x-ray detector based on this design and incorporating large area MCPs is discussed.

Development of the BIGMIC photon-counting detector for astronmomical applications

University College London and The Imperial College of Science, Technology & Medicine are together developing a new large area imaging photon counting system, BIGMIC, for use primarily on very large telescopes. This detector is designed for applications requiring the highest sensitivity and resolution such as, high dispersion Echelle spectroscopy. The system incorporates a specially designed 75mm active diameter image intensifier fibre- optically coupled to a fast scanning 770 x 576 pixel frame transfer CCD. Photon events at the intensifier output screen are centroided to 1/8 of a CCD pixel in both X and Y in order to sample adequately the point spread function of the intensifier, giving a data acquisition format of 6160 x 4608 sub-pixles. The area imaged onto the CCD is 61mm x 46mm with a data acquisition pixel size of 10 micrometers . A hardware windowing facility, built into the detector system, enables the astronomer to select a subset of the imaging area for data acquisition. This permits the user to match the system to specific applications; for example in a number of spectroscopic applications an essentially one dimensional image is required and a detector format of perhaps 6160 x 100 pixels could be utilized.