Fluorometers, simple

Fig. 8. Dependence of (A) corrected diffusion coefficient (D), (B) steady-state fluorescence intensity, and (C) corrected number of particles in the observation volume (N) of Alexa488-coupled IFABP with urea concentration. The diffusion coefficient and number of particles data shown here are corrected for the effect of viscosity and refractive indices of the urea solutions as described in text. For steady-state fluorescence data the protein was excited at 488 nm using a PTI Alphascan fluorometer (Photon Technology International, South Brunswick, New Jersey). Emission spectra at different urea concentrations were recorded between 500 and 600 nm. A baseline control containing only buffer was subtracted from each spectrum. The area of the corrected spectrum was then plotted against denaturant concentrations to obtain the unfolding transition of the protein. Urea data monitored by steady-state fluorescence were fitted to a simple two-state model. Other experimental conditions are the same as in Figure 6.

The first photoelectric fhiorimeter was described by Jette and West in 1928. The instrument, which used two photoemissive cells, was employed for studying the quantitative effects of electrolytes upon the fluorescence of a series of substances, including quinine sulfate [5], In 1935, Cohen provides a review of the first photoelectric fluorimeters developed until then and describes his own apparatus using a very simple scheme. With the latter he obtained a typical analytical calibration curve, thus confirming the findings of Desha [33], The sensitivity of these photoelectric instruments was limited, and as a result utilization of the photomultiplier tube, invented by Zworykin and Rajchman in 1939 [34], was an important step forward in the development of suitable and more sensitive fluorometers. The pulse fhiorimeter, which can be used for direct measurements of fluorescence decay times and polarization, was developed around 1950, and was initiated by the commercialization of an adequate photomultiplier [35]. 

Flow injection analysis (FIA) is a robust method for automating complex chemical analyses (Ruzicka and Hansen, 1988). It is relatively simple and can be adapted for use with a variety of detectors, including spectrophotometers, fluorometers, mass spectrometers, and electrochemical analyzers. It has been used on board ships to determine dissolved nutrients (Johnson et al., 1985) and trace metals (Sakamoto-Arnold and Johnson, 1987 Elrod et al., 1991). Unsegmented continuous flow analysis (CFA) systems based on the principles of FIA can operate in situ over the entire range of depths found in the ocean (Johnson et al., 1986a, 1989). 

Fig. I. Simple 90 -axis fluorometer as viewed from above H. radiation sixirce Ki and Fa. tillers S. shutters A. apertures or slits C. sample container...

Fluorescence detectors that use filters to select excitation and emission wavelengths are called filter fluorometers. This type of detector is the most sensitive, yet the simplest and least expensive. A diagram of this simple form of fluorescence detector is shown in Fig. 2. Usually, in order to enhance the fluorescence collected from the flow cell, lenses are employed along with filters. The lenses are positioned before the excitation filter and after the flow cell to focus and collect the light. 

Accurate estimates of all the kinetic coefficients in Eq. (1) or (2) are needed for the reaction in each direction, and at the same pH value, if reliable conclusions about mechanisms are to be reached. A sensitive method for measuring changes of reactant concentration is demanded because accurate estimates of v can only be made from progress curves that are linear for 30 sec or more, during which only a small fraction of the total reaction to equilibrium occurs. Moreover, Km values and dissociation constants of binary complexes of the coenzyme are often small (10 -10 M), and the equilibrium is usually unfavorable for NAD oxidation at pH 7.0 and in studies of product inhibition. Measurements of the fluorescence of the reduced coenzymes with a simple recording fluorometer provide a more sensitive analytical method than spectrophotometry 10,11). 

The analysis of simple mixtures of organic or inorganic compounds by fluorometry without any separation is often possible because of the versatility of fluorometers. In contrast to spectrophotometers, these instruments have two instrumental variables instead of one. The analysis of the following hypothetical mixture will illustrate the use of these variables. 

Anisotropic rotati onal diffusion has been more Nct sively studied using FD methods. In fact, the earliest reports on the anisotropic rotation of fluorophores concerned experiments performed using fixed-frequency phase-modulation fluorometers. At that time the phase-modulation instruments operated at only one or two fixed hrequendes. Hence, it was not possible to recover the anisotropy decay law. The experiments were performed by measuring the differential polarized phase angles as the temperature was varied. It is relatively simple to predict the maximum value of Ao) for known values of the lifetime and fundamental anisotropy. For an isotropic rotor, the predicted value of Aw is given by... 

Filter fluorometers provide a relatively simple, low-cost way of performing quantitative fluorescence analyses. As noted earlier, either absorption or interference filters are used to limit the wavelengths of the excitation and emitted radiation. Generally, fluorome-ters are compaet, rugged, and easy to use. 

Of the methods detailed in Table 11.1, FIA with gaseous diffusion and conductometric detection (Araujo et al., 2005) is the cheapest, and it consumes low amounts of reagents, uses simple materials, and allows rapid detection compared with the fluoromet-ric detection methods. 

Stopa, P. J. Mastromanolis, S. A. The use of blue-excitable nucleic-acid dyes for the detection of bacteria in well water using a simple field fluorometer and a flow cytometer. J. Microbiol. Methods 2001, 45, 143-153. 

---