Fluorescence scanner

In conventional chip experiments, fluorescence scanners are used for chip read-out. In the case of laser scanners, HeNe lasers are used as excitation sources and photomultiplier tubes as detectors, whereas CCD-based scanners use white light sources. The optical system can be confocal or non-confocal. Standard biochip experiments are performed using two fluorescent labels as... 

The background problem can be further overcome when using a surface-confined fluorescence excitation and detection scheme at a certain angle of incident light, total internal reflection (TIR) occurs at the interface of a dense (e.g. quartz) and less dense (e.g. water) medium. An evanescent wave is generated which penetrates into the less dense medium and decays exponentially. Optical detection of the binding event is restricted to the penetration depth of the evanescent field and thus to the surface-bound molecules. Fluorescence from unbound molecules in the bulk solution is not detected. In contrast to standard fluorescence scanners, which detect the fluorescence after hybridization, evanescent wave technology allows the measurement of real-time kinetics (www.zeptosens.com, www.affinity-sensors.com). 

Fluorescence scanners resolve fluorescence as a function of spatial coordinates in two dimensions for macroscopic objects such as electrophoresis gels, blots and chromatograms... 

Detect the fluorescent-labeled protein using a laser-based fluorescent scanner. An experimental example is shown in Fig. 3 see Note 23). 

Fig. 3. Detection of a synthesized protein by fluorescent labeling. Cell-free protein synthesis was carried out with or without the use of mRNA transcribed from a linearized expression done containing the p-gaiactosidase gene, and the synthesized protein was labeled by FluoroTect. The translational reaction mixtures were resolved by 12.5% SDS-PAGE. Detection of labeled protein was performed using a laser-based fluorescent scanner (FX pro, Bio-Rad, Hercules, CA). Lanes 1 and 2 represent negative control (absence of mRNA) and p-galactosidase, respectively.

Polycarbonate compact discs have also been used as supports for microarray development [112]. The microarray is generated using an inkjet applicator that employs an electric current to dispense monodisperse droplets containing antibodies onto the disk. The sensing reaction is based on competitive inhibition immunoassays using fluorescent antibodies and final readout is accomplished using a fluorescence scanner. 

Purification of the foll-length-labeled RNA can be performed by PAGE. Even small amount of the ligated product can be detected by fluorescent scanners, and detection of fluorescence from both dyes can be used to identify the correct band.4... 

Later, a four-color rotary confocal fluorescent scanner was designed to simultaneously detect 96 channels [977] and 384 channels [980]. The confocal scanner used to detect 384 channels on a microcapillary array electrophoresis (pCAE) chip is shown in Figure 7.3 [980],... 

FIGURE 7.3 The rotary confocal fluorescence scanner used to detect pCAE chip separations. Laser excitation at 488 nm (100 mW) is directed up through the hollow shaft of a computer-controlled stepper motor, deflected 1.0 cm off-axis by a rhomb prism, and focused on the electrophoresis lanes by a microscope objective. The stepper motor rotates the rhomb/objective assembly just under the lower surface of the microchip at five revo-lutions/s. Fluorescence is collected along the same path and spectrally, and is spatially filtered before impinging on the four-color confocal detector [980]. Reprinted with permission from the American Chemical Society. 

FIGURE 10.5 Electropherograms monitoring the dissociation of the Ab-TNB-Fl complex monitored at six consecutive detection points along the separation CE channel (see Figure 10.4 for locations) by using the rotary fluorescent scanner [285], Reprinted with permission from the American Chemical Society. 

The pre-hybridisation and hybridisation processes are run at the same temperature, i.e. 42 °C. As with global expression arrays, after hybridisation, the arrays are scanned using a fluorescent scanner (Figure 3). 

Fig. 4.16 Fluorescence scanner images of a functionalized microtiter plate with a library of 21 SAMs configured into an array (for library composition see Fig. 4.13), before and after exposure to a 10 4 M acetonitrile solution of Cu(C104)2, respectively 58. Reproduced with permission...

The examples of small combinatorial libraries presented in this chapter were all fabricated manually, but automation of the process could be implemented using for example the already developed techniques for microarray fabrication, microspotter robots for fabrication, and fluorescence scanner for analysis. We hope that this chapter constitutes a clear explanation of the fabrication of combinatorial libraries of fluorescent self assembled monolayers and their applications. 

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