Read-out systems

As stated earlier, activation of endothelial cells by pro-inflammatory stimuli leads to the expression of cell adhesion molecules and cytokines such as IL-6 and IL-8. The expression and hence modulation of surface expressed adhesion molecules by e.g. targeted delivery of inhibitors of NFkB, can be measured using flow cytometric analysis or whole cell ELISA techniques. Cytokine production can be measured in the supernatant of cultured cells or in biological fluids. Furthermore, competitive or quantitative RT-PCR analysis of mRNA levels of cell adhesion molecules or cytokines, allows the transcriptional activity of the genes of interest to be estimated. 

To investigate the effects of drugs on NFkB activation at the molecular level, the Electric Mobility Shift Assay (EMSA) is a useful read-out system. With this technique the nuclear localization of this transcription factor following activation and subsequent translocation can 

7 Vascular Endothelium in Inflamed Tissue as a Target for Site Selective Delivery of Drugs 

EMSA assays can also be exploited to measure STAT nuclear localization, which is, similar to NFkB localization, a measure of STAT activity. Determination of JAK phosphorylation is carried out by immunoprecipitation of the JAK proteins from cell lysates, followed by SDS-PAGE electrophoresis, immunoblotting with antiphosphotyrosine antibody and JAK-specific antibody re-probing [99]. 

Many animal models have been described in which inflammation is induced and activation of the endothehum occurs. Here, some disease-specific models, in addition to some general inflammatory experimental models will be summarized. 

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The read-out systems available include meters, chart recorders, and digital display meters have now been virtually superseded by the alternative methods of data presentation. 

ATOM RESERVOIR MONOCHROMATOR PHOTOMULTIPLIER READ OUT SYSTEM... 

Photomultipliers are generally used to convert the spectral radiation to an electrical current and often phase-sensitive lock-in amplifiers are used to amplify the resulting current. AES and AFS require similar read-out systems because both methods are measuring small signals. The difficulty associated with both these methods is the separation of the signal for the atomic transition of interest from the background radiation emitted by excited molecular species produced in the atom reservoir. AFS phase locks the amplifier detection circuit to the modulation frequency of the spectral source. Modulation of the source is also used in AAS. 

After suitable amplification, the signal may be read out using a variety of different approaches. Older instruments used an analogue meter as a means of read-out. Considerable advances have been made in read-out systems... 

Over the years a panel of methods has been published for testing different properties of the SC barrier (Table 37.1). The combination of tests, employing the application of chemical probes to the skin and the measurement of skin responses with new bioengineering read-out systems allows to measure changes in barrier properties with high sensitivity and reliability. A subjective selection of skin barrier tests will be discussed further in this chapter. 

Where the influence of affinity or kinetic behavior is unclear or less important than the precise epitope recognized, for example, in virus or cytokine neutralization, or in receptor triggering, fast read-out systems independent of binding tests are required. 

A fundamental contribution concerning the optimization of filtering for position sensitive read-out system is made by V. Radeka of Brookhaven National Laboratory... 

As indicated in Fig. 5, the signal is spread out over several elements of the read-out electrode. The role of a position sensitive read-out system is to determine the centroid of the signal distribution. 

The technique was first introduced in 1968, as a read-out system for a two-dimensional MWPC for high energy physics experiments Basically, the signal induced in the read-out strips propagates towards both sides of the delay-line, and the difference in time of arrival at both ends is a measure for the position of the avalanche. 

Although the local count rate is limited by the space charge effect, as we have seen before, it is the read-out system which will be the limiting component of the system as a whole. Certainly with synchrotron radiation as a source, but already with a rotating anode X-ray generator, area detector systems will be saturated quickly. In addition, the demands concerning the data acquisition system become even more stringent. 

In many applications full two-dimensional information is necessary, and thus fast read-out systems have to be provided. The various techniques, which have been discussed before, have been applied with area detectors. The delay-line read-out has been used with two-dimensional detectors in synchrotron radiation laboratories. At the DORIS-ring in Hamburg an area detector with a 1 mm anode wire distance and a total area of 200 mm x 200 mm is currently in use for measurements of muscle diffraction patterns and for X-ray crystallography The spatial resolution is about (2,5x2,5)mm FWHM. 

A new type of imaging devices has been introduced a decade ago These devices consist of an array of photo-sensitive diodes, coupled to a CCD read-out system. CCD s are based upon the transfer of charge packets in a repetitive metal-oxid°-silicon (MOS) structure (see Fig. 20). The charges in the elements of a CCD can be photo-generated, e.g. by the absorption of X-rays in the substrate, and then shifted out electronically to a single output. In a way CCD s operate as the solid state selfscanning equivalent of a vidicon-tube. 

Another kind of linear solid state position sensitive detectors are the Photo-Diode-Arrays (PDA s), which are different from CCD s. A PDA consists of an array of separate photodiodes, each with an associated capacitance and a multiplexing read-out system (see Fig. 21). The charges collected in each cell are simply switched to the output, one by one. Unlike in the case of CCD s, the photosensitive elements are separated completely from the transfer circuity. 

This read-out system makes it possible for more than one portion of the long template molecule to be read, by different ribosomes, at a given time and, in fact, in cells which are actively synthesizing proteins, it is possible to isolate, by suitably gentle techniques, such template RNA molecules with several ribosomes attached to different portions of them. Such a cluster of ribosomes held together by a template is called a polysome . This simultaneous read-out enables many molecules of the same protein to be made simultaneously in response to cellular demand. 

A further increase in speed of data taking is expected by segmentation of detectors, i.e. each detector segment has its own read-out system. A linear position-sensitive detector develop recently at EMBL consists of 128 anode wires, with each of them acting as an individual counter. The integral count rate is 10 MHz of statistical events. A similar development has been started for area counters. The new diffractometer for X-ray resonance scattering in HASYLAB is equipped with three area counters (Fig. 5). This system can handle nearly a million statical events per second... 

The present modern positon-sensitive counters in general cope with the scattering intensity produced at the present synchrotron radiation sources. As an increase of the brilliance of synchrotron radiation by 1000 to 10000 is expected with planned electron storage rings, considerable effort has to go into the development of area detectors and their read-out systems. 

Fast-responding sensors will pose no significant problems for the read-out system. 

Sources of error in the sample preparation should be recognized and interferences controlled. However, each analysis involves random (statistical) errors, and the whole error is the sum of cumulative errors at each stage of an analytical procedure. A number of effects contribute to the uncertainty of the final signal displayed on the readout system. In the measurement stage various sources of interference are fluctuations in radiation source signal, photomultiplier shot noise , electronic noise , flame fluctuations, nebuliza-tion and atomization noise , inaccuracies in the read-out system, and interelement interferences. 

FIGURE 16.15 (a) Typical cross-sectional structure of an optical disk and (b) the intensity profile of the read-out system in binary code as a function of time (t). ffere, T is the transparent polymer support, R is the reflective metal layer, PC is the protective coating, and P represents a depression created for information storage. (Reproduced with permission from HUthig and Wepf Verlag.)... 

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