Fluorescence scanners, detection

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). 

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.

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. 

Fluorescence scanners and fluorescence microscopes, which detect fluorescence as a function of spatial coordinates in two or three dimensions for microscopic objects. 

Dry the slide by purging with Ar gas and detect bound proteins using a fluorescence scanner (see Fig. 4). 

FIGURE 46.6 The Berkeley rotary confocal fluorescence scanner developed by James R. Scherer. Laser excitation is directed up through the hollow drive shaft of a stepper motor, deflected off axis by a rhomb prism, and is then focused with a microscope objective, tracing a 1-cm radius circular detection path on the chip. Fluorescence is collected along the same path and then spatially and spectrally filtered by a four-color PMT array. The inverted nature of this system allows easy access to the upper surface of a jjiCAF device for electrical, pneumatic, and sample interfacing. 

Since the freedom of design for microfluidics in various detections, the confocal LIF was restructured as rotary confocal fluorescence scanner to simultaneously detect numerous detection points to match the high-throughput nucleic acid analysis [5]. 

Microarrays are used for detection and quantification of the target gene using a DNA chip on which known DNA molecules are fixed. Complementary DNAs (cDNA) are reverse transcribed from RNAs isolated from culture samples, and the DNA extracted from environmental samples is hybridised to the nucleotide probes on the DNA chip. The signal intensities of these DNA-probe hybridised molecules are then detected using a fluorescence scanner. 

The major difference in the typing of the STR loci is the ability to include an internal size standard if the detection device used has multicolor capability. Under the TWG-DAM Guidelines forensic samples are to be placed adjacent to an allele ladder, as seen in Fig. 7 (PCR-STR). Since the Beckman Genomyx SC only has two filters (fluorescein and TMR) an internal ladder could not be used, so the adjacent ladder format is used. In this situation there is no special preparation for detection. When the four-color Hitachi FMBIO II Fluorescent Scanner, ABI Prism 377 or 310 is used, an internal standard is used to size the DNA fragements. As part of the electrophoresis setup a ROX ladder is added to PE Biosystems amplified products while a CRX ladder is added to Promega kits. (See Figure 8 for example.) Amplified products including the... 

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