Fluorescence fluorimeters

Fig. 7. Fluorescence polarization (FP). (a) The formation of the large FITC—protein A—IgG complex which leads to a net increase in plane polarized light transmitted from the solution. Molecular weights of the protein A-FITC, IgG, and complex are ca 43,000, 150,000, and 343,000, respectively, (b) Detection of IgG by fluorescence polarization immunoassay using A, a laboratory fluorimeter where (O) represents AP = change in polarization, and B, a portable detection unit where (D) is —fiV = change in voltage (27). The field detector proved to be more sensitive than the fluorimeter.

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... 

Procedure. Measure the fluorescence of each of the above solutions at 445 nm, using that containing 62.0 mL of the dilute quinine solution as standard for the fluorimeter. Use LF2 or an equivalent primary filter (/cx = 350 nm) and gelatin as the secondary filter if using a simple fluorimeter. 

Once the analyte has been separated from the matrix in LC, the best approach to the detection of the molecule must be determined. This section will discuss the detection techniques of ultraviolet/visible (UVA IS), fluorescence (FL), and electrochemical (EC) detection, with MS being addressed separately in Section 4.2. When deciding on the most appropriate detector for an LC separation, the appropriate chemical data on the analyte should be collected by using a spectrophotometer, fluorimeter, and potentiometer. 

For FL detection, maximum emission and excitation wavelengths are determined using a fluorimeter. Stoev used fluorescence detection to analyze for closantel (excitation at 335 nm, emission at 510 nm) residues in animal tissue. 

Colored and fluorescent dyes have the advantage of being relatively cheap and easy to use. Standard procedures are available for detection of the dyes using colorimeters and fluorimeters. Some of these instruments can be used in the field to analyze samples as they are collected following exposure to the dyes. Fluorescein has been widely used for studying spray deposition within and outside canopies. ... 

For the investigation of triplet state properties a laser flash photolysis apparatus was used. The excitation source was a Lambda Physik 1 M 50A nitrogen laser which furnished pulses of 3.5 ns half-width and 2 mJ energy. The fluorescence decay times were measured with the phase fluorimeter developed by Hauser et al. (11). 

Fluorescence Lifetimes. Fluorescence lifetimes were determined by the phase shift method, utilizing a previously-described phase fluorimeter. The emission from an argon laser was frequency doubled to provide a 257 nm band for excitation. Fluorescence lifetimes of anisole and polymer 1 in dichloro-methane solution were 2.2 and 1.4 nsec, respectively. Fluorescence lifetimes of polymer films decreased monotonically with increasing DHB concentration from 1.8 (0) to 0.7 nsec (9.2 x 10 3 MDHB). Since fluorescence lifetimes (in contrast to fluorescence intensities) are unaffected by absorption effects of the stabilizer, these results provide direct evidence in support of the intensity measurements for RET from polymer to stabilizer. 

On continued excitation in the phase fluorimeter, the fluorescence lifetime of polymer 1 films also decreased with time. The lifetime decrease was exponential with an average loss constant of 8.2 1.2 x 10 lf sec-1 (1.5 pm thick film) from measurements at different sites on the film. These findings constitute direct evidence for RET from the polymer to a photoproduct(s) in support of the fluorescence intensity measurements. 

Fluorescence Studies. Fluorescence spectra of films on glass plates were obtained with a Perkin-Elmer MPF-3 spectro-fluorimeter. A previously-described phase fluorimeter was utilized for fluorescence lifetime determinations. 

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]. 

Luminescence instrument LS-3B luminescence instrument LS-5B Accessories low flow cell, cell holders, bioluminescence spectroscopy, fluorescence spectro scopy, recorder/printers, low-temperature luminescence, fluorescence plate reader, polarization accessory, microfilm fluorimeter LS-2B... 

Measurement of fluorescence intensity can be used for quantitative analysis of fluorescent compounds where the intensity of fluorescence is proportional to the concentration of the compound. Because of their high sensitivity and selectivity, analytical techniques based on fluorescence detection are commonly used. If a target compound is fluorescent then direct detection of the fluorescence emitted is possible using a fluorimeter (Figure 4.7). 

Figure 8.11. Diffuse reflectance absorption spectra of a strongly fluorescent sample (1,6-diphenylhexatriene adsorbed on porous alumina) (a) conventional measurement w ith monochromatic irradiation and detection via an integrating sphere (b) measurement in a fluorimeter with two monochromators. Reaction spectra during Irons - cis photoisomerization are also given (adapted from Ref. 26).

The first measurement we make when starting a fluorescence study is not usually a fluorescence measurement at all but the determination of the sample s absorption spectrum. Dual-beam differential spectrophotometers which can record up to 3 absorbance units with a spectral range of 200-1100 nm are now readily available at low cost in comparison to fluorimeters. The wide spectral response of silicon photodiode detectors has made them preeminent over photomultipliers in this area with scan speeds of a few tens of seconds over the whole spectral range being achieved, even without the use of diode array detection. 

It will be seen that, as in the case of the LED, control of the bias voltage gives simple modulation of the laser output intensity. This is particularly useful in phase-modulation fluorometry. However, a measure of the late awareness of the advantages of IR techniques in fluorescence is that only recently has this approach been applied to the study of aromatic fluorophores. Thompson et al.(51) have combined modulated diode laser excitation at 670 and 791 nm with a commercial fluorimeter in order to measure the fluorescence lifetimes of some common carbocyanine dyes. Modulation frequencies up to 300 MHz were used in conjunction with a Hamamatsu R928 photomultipler for detecting the fluorescence. Figure 12.18 shows typical phase-modulation data taken from their work, the form of the frequency response curves is as shown in Figure 12.2 which describes the response to a monoexponential fluorescence decay. 

Fig. 2. Parameters affecting the efficiency of energy transfer. (A) Overlay of FITC emission spectrum and PE absorbance spectrum normalized to maximum fluorescence intensity and maximum optical density, respectively. FITC fluorescence intensity was measured as a function of emissions wavelength using a fluorimeter with an excitation wavelength of 488 nm. PE optical density was measured as a function of wavelength using a spectrophotometer. (B) Schematic representation of energy absorption and the possible pathways for the subsequent energy release (abbreviations as in the text).

Compounds that interfere with the detection mechanism of the HTS assay wiU, in many cases, be detected as highly potent actives [31]. One example would be compounds that intrinsically emit or absorb light at the wavelengths used in a fluorescence-based assay such as fluorescence resonance energy transfer (FRET). In an HTS screen using a fluorescence-based assay at Wyeth, 1.2% of the samples tested showed not just high fluorescence but the maximum possible initial (time 0) reading on the fluorimeter. 

Fluorimeters use a photomultiplier (or PM) tube that is positioned perpendicular to the incident light beam to detect fluorescence emission by a sample. The photomultiplier has a light-sensitive surface (the photocathode) which is maintained at a negative potential of about 950-1500 volts. So-called photoelectrons are released from photocathode upon light absorption, and they are... 

Surface fluorescence of NADH/NADPH can be recorded continuously with a DC fluorimeter and correlated with changes in experimental conditions. A mercury arc lamp (with a 340-375 nm filter in front) is used as a hght source for fluorescence excitation. The fluorescence response of reduced NADH/NADPH was measured at 450-510 nm. The DC fluorimeter and the Hg arc lamp are connected to the kidney by a trifurcated fiber optics light guide. NADH/NADPH fluorescence emission can be corrected for changes in tissue opacity by a 1 1 subtraction of reflectance changes at 340-375 nm from the fluorescence. To determine NADH/NADPH redox state of the total surface area of kidney cortex and to evaluate whether certain areas were insufficiently perfused, fluorescence photographs of the total surface area were taken. The study demonstrated that the surface fluorescence method is simple and provides specific information about the mitochondrial oxidation-reduction state. 

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