Fluorescence standards

Nonradiative reiaxation and quenching processes wiii aiso affect the quantum yieid of fluorescence, ( )p = /cj /(/cj + Rsiative measurements of fluorescence quantum yieid at different quencher concentrations are easiiy made in steady state measurements absoiute measurements (to detemrine /cpjj ) are most easiiy obtained by comparisons of steady state fluorescence intensity with a fluorescence standard. The usefuiness of this situation for transient studies... 

Functions of Standards. Fluorescent standards can be used for three basic functions calibration, standardization, and measurement method assessment. In calibration, the standard is used to check or calibrate Instrument characteristics and perturbations on true spectra. For standardization, standards are used to determine the function that relates chemical concentration to Instrument response. This latter use has been expanded from pure materials to quite complex standards that are carried through the total chemical measurement process (10). These more complex standards are now used to assess the precision and accuracy of measurement procedures. 

Requirements of Standards. Standards used In the calibration of mlcrospectrofluorometrlc Instrumentation must meet more stringent requirements than those used In macromeasurements. The effect of Increased excitation flux as well as spatial effects under magnification make many macromeasurement standards unsatisfactory for micromeasurements. Following are some of the requirements for fluorescence standards that are strongly Influenced by a change from the macro- to the micro-environment. 

We see then that the relative fluorescence quantum yield can be determined by measuring the areas under the fluorescence bands of the sample and the fluorescent standard. However, these spectra must be corrected before their true areas can be determined. Several factors are responsible for this. The most important of these are the phototube and monochromator responses. For most phototubes the maximum response occurs within a limited wavelength range, falling off rather sharply in some cases at the short-and long-wavelength ends. This is illustrated in Figure 2.14. Similarly,... 

If the refractive indices of the solvents used for the sample and the fluorescence standard are not the same, a further correction must be made. For example, quinine sulfate in 0.1 N H2S04 (Or = 0.5) is commonly used as a fluorescence standard. If the fluorescence of the sample whose relative quantum yield is desired is determined in benzene, a correction factor of 27% must be applied in determining the relative areas under the fluorescence bands. If ethanol is used, this correction is only 5.5%. 

In most cases, the linear absorption is measured with standard spectrometers, and the fluorescence properties are obtained with commercially available spectrofluo-rometers using reference samples with well-known <1>F for calibration of the fluorescence quantum yield. In the ultraviolet and visible range, there are many well-known fluorescence quantum yield standards. Anthracene in ethanol (Cresyl Violet in methanol (commonly used reference samples for wavelengths of 350-650 nm. For wavelengths longer than 650 nm, there is a lack of fluorescence references. Recently, a photochemically stable, D-ji-D polymethine molecule has been proposed as a fluorescence standard near 800 nm [57]. This molecule, PD 2631 (chemical structure shown in Fig. 5) in ethanol, has linear absorption and fluorescence spectra of the reference PD 2631 in ethanol to... 

Although several types of fluorescent beads were proposed as a microscopic fluorescence standard 30 years ago,2 beads have not been used as a proteinembedding matrix for routine IHC on FFPE tissue. We recently tested primary coated beads ( Dynabeads, Dynal, New York) that are coated with a goat anti-mouse antibody on the surface of the beads. In the first experiment, a monoclonal antibody to cytokeratin 7 (DAKO, 50pL/34.5pg) was bound to the beads by incubating with the beads (150 pL at a concentration of 109 beads/1 pL) at 4°C in a cold room with an automatic shaker for overnight. Incubation was followed by three phosphate-buffered saline (PBS) washes,... 

Resch-Genger U, Hoffmann K, Nietfeld W, Engel A, Neukammer J, Nitschke R, Ebert B, Macdonald R (2005) How to improve quality assurance in fluorometry fluorescence-inherent sources of error and suited fluorescence standards. J Fluoresc 15 337-362... 

In frequency-domain FLIM, the optics and detection system (MCP image intensifier and slow scan CCD camera) are similar to that of time-domain FLIM, except for the light source, which consists of a CW laser and an acousto-optical modulator instead of a pulsed laser. The principle of lifetime measurement is the same as that described in Chapter 6 (Section 6.2.3.1). The phase shift and modulation depth are measured relative to a known fluorescence standard or to scattering of the excitation light. There are two possible modes of detection heterodyne and homodyne detection. 

A few materials are given that can be used as primary fluorescence standards in the adsorbed state, because they have high yields, show little tendency of aggregation provided the surface coverage is low, and are photochemically stable ... 

Enzymic hydrolysis (25-40°C) at the heterosidic bond of the chromogenic substrates was followed either continuously (via formation of 2 -chloro,4 -nitrophenol) at pH 5.5 (O.D. 405 nm) or discontinuously (4-methylumbel-liferone fluorescence at pH 10, emission at A > 435, excitation at A366 nm). Reaction rates were calculated from the linear increase of O.D. (em = 9000 M-1cm-1) or fluorescence (standardization with 4-methylumbelliferone) versus time. Alternatively, an HPLC method was used to follow the formation of chromophoric reaction products, phenols and glycosides (1). Concentrations were calculated from peak heights after appropriate standardization. 

The measured intensities of TPEF as a function of the spectral bandwidth, for GDD-compensated and TL pulses, are shown in Figure 8.2. The experiments were performed with the same average power at the sample (2.3 mW) for all bandwidths, using a red fluorescence standard slide (Chroma Technologies). We found that in the case of TL pulses, obtained via MIIPS compensation, the magnitude of TPEF... 

The measurement of quantum yield is a more complicated process. Before these measurements can be made, the instrument must be calibrated. A thermopile or chemical actinometer may be used to measure the absolute intensity of incident light on the sample. Alternatively, quantum yields may be measured relative to some accepted standard. Two commonly used fluorescence standards are quinine sulfate in 0.5 M H2S04 (jQ = 0.70) and fluorescein in 0.1 M NaOH (f9 = 0.93). The quantum yield of the unknown, Q, is then calculated by Equation 5.7. 

Overwhelmingly, the published research on Raman-based biofluid analysis uses excitation in the near-infrared regime in order to reduce fluorescence. Standard linewidth-narrowed diode lasers at 785 or 830 nm are most common. As noted in Chap. 1, several hundred milliwatts of multiple-spatial mode light can routinely be obtained from diode lasers, making them economical choices... 

Electronic Spectrum. Acetone is the simplest ketone and thus has been one of the most thoroughly studied molecules. The it n absorption spectrum extends from 350 nm and reaches a maximum near 270 nm (125,175). There is some structure observable below 295 nm, but no vibrational and rotational analysis has been possible. The fluorescence emission spectrum starts at about 380 nm and continues to longer wavelengths (149). The overlap between the absorption and the fluorescence spectra is very poor, and the 0-0 band has been estimated to be at - 330 nm (87 kcal/mol). The absorption spectra, emission spectra, and quantum yields of fluorescence of acetone and its symmetrically methylated derivatives in the gas phase havbe been summarized recently (101). The total fluorescence quantum yield from vibrationally relaxed acetone has been measured to be 2.1 x 10 j (105,106), and the measurements for other ketones and aldehydes are based on this fluorescence standard. The phosphorescence quantum yield is -0.019 at 313 nm (105). 

Fluorescence quantum efficiencies of several solid materials have been measured by photoacoustic spectroscopy." The photophysics of quantities for some common fluorescence standards have been made with some accuracy the influence of refractive index corrections on yield and lifetimes are discussed, 9,10-diphenylanthracene, quinine bisulphate, and 2-aminopyridine being the selected examples. Correction for inner filter effects in fluorescence spectroscopy have been proposed. ... 

The quantum yields of fluorescence and phosphorescence, 4>f and d>p, may be determined experimentally by means of a fluorescent standard such as a rhodamine B solution whose independent of the exciting wavelength within a wide range. Lifetimes rp and rp are also experimentally accessible through time-resolved fluorescence measurements (phase method or single-photon counting) or by measuring the time dependence of phosphorescence. (Cf. Rabek, 1982.) In Table 5.2 the observable quantities and their relationship to rate constants are collected. 

The correction of systematic phase errors in frequency domain spectroscopy can be achieved by use of a fluorophore of known lifetime as standard. It has been pointed out that a simple scattering solution can be used as standard. This ingenious suggestion very conveniently dispenses with the need for a fluorescent standard with a previously-determined lifetime value. Phase noise, another troublesome factor encountered in frequency domain fluorimetry, can be eliminated by use of a differential method. 

Fluorescene intensity is given semiquantitatively according to a fluorescence standard. A = without hyperkeratoses B = with intensive hyperkeratotic crusting. 

The Perkin-Ulmcr Ciorporation offers a set of six fluorescence standards dissolved in a plastic matrix to give stable solid block.s that can he used indefinitely without special. storage. With these, the instrument is easily. standardized for the wavelength region to be used for the analysis. 

In principle the wavelength of irradiation and excitation are the same. The excitaticm light source is used to cause the photoreaction. By these means monochromatic irradiation is used automatically. A typical cell compartment is given in Fig. 4.22. The sample is stirred and temperature controlled. Three positions are mounted tilted at 45 relative to the optical axis of the excitation light beam. One position is for the cell with the solvent, one for the fluorescence standard, and the third for the sample itself. These three positions... 

Is controlled by a split-beam and a photodiode. The 45" tilted bar contains three positions (1) for a fluorescence standard (S), (2) for the sample (R), (3) for a reference blank (v). The sample can be stirred. The cell holder is temperature controlled. The irradiation source is either a... 

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