Fluorescent detection, instrument excitation source

Precision When the analyte s concentration is well above the detection limit, the relative standard deviation for fluorescence is usually 0.5-2%. The limiting instrumental factor affecting precision is the stability of the excitation source. The precision for phosphorescence is often limited by reproducibility in preparing samples for analysis, with relative standard deviations of 5-10% being common. 

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. 

Phosphorescence spectra (uncorrected, front face) were recorded on a Perkin-Elmer LS-5 fluorescence spectrometer using a pulsed excitation source ( 10 ps) and gated detection. The instrument was controlled by a P-E 3600 data station. The samples were typically excited at 313 nm using the instrument s monochromator and an additional interference filter. Excitation and emission bandpasses were 2 nm. Typically the emission spectra were recorded using a 50 ps delay following excitation and a 20 ps gate. The samples were contained in cells made of 3x7 mm2 Suprasil tubing, under a continuous stream of N2, 02 or 02/N2 mixtures of known composition. 

The time resolution of the electronics in a single photon counting system can be better than 50 ps. A problem arises because of the inherent dispersion in electron transit times in the photomultiplier used to detect fluorescence, which are typically 0.1—0.5 ns. Although this does not preclude measurements of sub-nanosecond lifetimes, the lifetimes must be deconvoluted from the decay profile by mathematical methods [50, 51]. The effects of the laser pulsewidth and the instrument resolution combine to give an overall system response, L(f). This can be determined experimentally by observing the profile of scattered light from the excitation source. If the true fluorescence profile is given by F(f) then the... 

Finally, the cell pellet is suspended in 5 00 pi FACS buffer and analysed for doxorubicin fluorescence using a FACS instrument (here FACS Vantage microflow cytometer, Beckton Dickinson) using the 488 nm line of an air cooled argon laser as the excitation source. Fluorescence from cell associated doxorubicin is detected using a 550 nm long pass emission filter. 

Laser excitation for fluorescence detection has received much research interest, but as of yet there is no commercially available instrument. Fluorescence intensity increases with excitation intensity, and it is generally assumed that laser excitation would then offer improved limits of detection. However, as Yeung and Synovec have shown, various types of light scattering, luminescence from the flow cell walls, and emission from impurities in the solvent all increase with source intensity as well, yielding no net improvement in signal-to-noise ratio (53). Where laser excited fluorescence may prove useful is for the design of fluorescence detectors for microbore packed and open tubular LC columns, where the laser source can be focused to a small illuminated volume for on-column detection. 

The x-ray fluorescence spectrometer consists of three main parts the excitation source, the specimen presentation apparatus, and the x-ray spectrometer. The function of the excitation source is to excite the characteristic x-rays in the specimen via the x-ray fluorescence process. The specimen presentation apparatus holds the specimen in a precisely defined position during analysis and provides for introduction and removal of the specimen from the excitation position. The x-ray spectrometer is responsible for separating and counting the x-rays of various wavelengths or energies emitted by the specimen. In this book the term x-ray spectrometer denotes the collection of components used to disperse, detect, count, and display the spectrum of x-ray photons emitted by the specimen. When referring to the entire instrument, including excitation source, sample presentation apparatus, and x-ray spectrometer, the term x-ray fluorescence spectrometer will be used. In this latter sense the term x-ray fluorescence analyzer is sometimes encountered in the literature. 

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