Fluorescence filter instruments

Photomultipliers are appreciably more sensitive sensors than the eye in their response to line or continuum sources. Monochromators are fitted to the light beam in order to be able to operate as substance-speciflcally as possible [5]. Additional filter combinations (monochromatic and cut-off filters) are needed for the measurement of fluorescence. Appropriate instruments are not only suitable for the qualitative detection of separated substances (scanning absorption or fluorescence along the chromatogram) but also for characterization of the substance (recording of spectra in addition to hR and for quantitative determinations. 

This paper reports on research involved the design, construction, and evaluation of a portable instrument, a "luminoscope", for detecting skin contamination by coal tars via induced fluorescence. The instrument has been used in the laboratory to measure the fluorescence of various coal tars and recycle solvents from liquefaction processes spotted on filter paper on rat and on hamster skin. The practical use of the devices in field test measurements to monitor skin contamination of workers at coal gasifier is discussed. The paper also discusses the practicality and usefulness of the luminescence method for detecting skin contamination. 

Fluorescence spectrum A plot of fluorescence intensity versus wavelength in which the excitation wavelength is held constant. Fluorometer A filter instrument for quantitative fluorescence measurements. 

An instrument for measuring fluorescence that uses filters to select the excitation and emission wavelengths. 

Instruments for the measurement of fluorescence are known as fluorimeters or spectrofluorimeters. The essential parts of a simple fluorimeter are shown in Fig. 18.1. The light from a mercury-vapour lamp (or other source of ultraviolet light) is passed through a condensing lens, a primary filter (to permit the light band required for excitation to pass), a sample container, a secondary filter (selected to absorb the primary radiant energy but transmit the fluorescent... 

The accuracy with which a system can measure lifetimes depends on a number of different factors including calibration of the instrument, the number of detected photons and also the efficiency of the analysis routines. In addition, sources of background and scattered light should be eliminated. Emission filters should be chosen with great care to make sure that no scattered laser light reaches the detector. Detection of scattered excitation light results in a spurious fast component in the decay and complicates the interpretation of the data. The choice of emission filters is much more critical in FLIM than in conventional fluorescence intensity imaging methods. 

Fluorescent detection technology applicable to biochips is evolving rapidly, resulting in detection instruments that are more powerful, user-friendly, and less expensive. Most systems employ photomultiplier tube (PMT) technology in conjunction with multiple colors, lasers, and a variety of filters. It is essentially a fluorescent microscope that... 

The basic instrumentation used for spectrometric measurements has already been described in the previous chapter (p. 277). Methods of excitation, monochromators and detectors used in atomic emission and absorption techniques are included in Table 8.1. Sources of radiation physically separated from the sample are required for atomic absorption, atomic fluorescence and X-ray fluorescence spectrometry (cf. molecular absorption spectrometry), whereas in flame photometry, arc/spark and plasma emission techniques, the sample is excited directly by thermal means. Diffraction gratings or prism monochromators are used for dispersion in all the techniques including X-ray fluorescence where a single crystal of appropriate lattice dimensions acts as a grating. Atomic fluorescence spectra are sufficiently simple to allow the use of an interference filter in many instances. Photomultiplier detectors are used in every technique except X-ray fluorescence where proportional counting or scintillation devices are employed. Photographic recording of a complete spectrum facilitates qualitative analysis by optical emission spectrometry, but is now rarely used. 

In the laboratory, solutions of analytes that fluoresce are tested by measuring the intensity of the light emitted. The instrument for measuring fluorescence intensity is called a fluorometer. Inexpensive instruments used for routine work utilize absorption filters similar to what was described previously for absorption spectrophotometers (see Figure 8.2 and accompanying discussion) and are called filter fluorometers. Two such filters are needed—one to isolate the wavelength from the source to be absorbed, the wavelength... 

---