Fluorescence detection sources

Better detection limits are obtained using fluorescence, particularly when using a laser as an excitation source. When using fluorescence detection, a small portion of the capillary s protective coating is removed and the laser beam is focused on the inner portion of the capillary tubing. Emission is measured at an angle of 90° to the laser. Because the laser provides an intense source of radiation that can be focused to a narrow spot, detection limits are as low as 10 M. 

First, the above-mentioned sensors have major drawbacks, as the detection and recognition event is a function of the nature and characteristics of the side chains, and the side chain functionalization of the CP requires advanced synthesis and extensive purification of numerous monomeric and polymeric derivatives. Second, this generation of sensors primarily employed optical absorption as the source for detection, resulting in lower sensitivity when compared with other sensing systems for biological processes. However, the use of fluorescence detection within these sensing systems could justify continued development. More recent examples include a fluorescent polythiophene derivative with carbohydrate functionalized side chains for the detection of different bacteria [15] and novel synthesis schemes for ligand-functionalization of polythiophenes [16]. 

Finally, we note that future instrument for lifetime-based sensing and imaging can be based on laser diode light sources. At present it is desirable to develop specific probes which can be excited from 630 to 780 nm, the usual range of laser diodes. The use of such probes will allow us to avoid the use of complex laser sources, which should result in the expanded use of fluorescence detection in the chemical and biomedical sensors. 

In addition to an intense source and a well matched monochromator/mirror system, detectors require optimisation. For protein XAS, it is now well established that fluorescence detection is the preferred mode of detection Multi-detector... 

Fluorescein (703) (Acid Yellow 73 C.I. 45350) is possibly the best known xanthene dye. The sodium salt is soluble in water to which it imparts an intense yellow-green fluorescence, detectable even at a dilution of 0.02 p.p.m. under UV irradiation. This property leads to the use of fluorescein as a location marker for aircraft lost at sea, as a tracer for the detection of a source of contamination in drinking water, and in a number of related situations. The use of fluorescein to detect abrasions of the cornea is also based on its fluorescence. 

The absorption and emission maxima from this table will provide clues to the spectral ranges that are useful for excitation and for fluorescence detection with a particular fluorochrome. However, the absorption and emission spectra have breadth, with slopes and shoulders and secondary peaks (see Fig. 5.6). With efficient fluorochromes, excitation and fluorescence detection at wavelengths distant from the maxima may be possible. Therefore, inspection of the full, detailed spectra is necessary to get the full story. In addition, spectra may shift in different chemical environments (this will explain why maxima vary in different reference tables from different sources). Values in this table are derived primarily from the Molecular Probes Handbook and the article by Alan Waggoner (Chapter 12) in Melamed et al. 

Finally, the use of CCD cameras as detectors in combination with short-pulsed light sources enables not only spatially resolved imaging, but also a time-resolved fluorescence detection. Time resolution offers two different new alternatives ... 

The LED has also been used as the excitation source for fluorescent detection. For instance, two LEDs (red and blue) were used to detect FTTC-labeled amino acids on a PMMA chip. The blue LED was used for excitation and the red LED for background noise compensation, leading to a four-fold S/N enhancement [681], Moreover, blue LEDs (470 nm) were used to detect various dyes, such as... 

In fluorescent detection, an argon ion laser is usually used for excitation. Suggest one fluorophore that is compatible with this source, and describe why there are two associated wavelengths. (3 marks)... 

What are the light source and detector used in the microchip with integrated fluorescent detection [692] (2 marks)... 

For EXAFS and particularly for XANES, data analysis is complex. The oscillation frequency/bond distance dependence means that extensive use is made of Fourier transform analysis. Most applications to date have been in the EXAFS region. In order to acquire sufficiently strong signals in a reasonable time, use has to be made of high-intensity photon fluxes, which are available at synchrotron facilities. These provide a broad-band tuneable source of high-intensity radiation, but the reduced number of facilities limits widespread dissemination of the technique. Reflection (fluorescent detection) mode is usually preferred to transmission. Experiments can be conducted in any phase, and the probing of electrode surfaces in situ is an important application. 

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