Fluorescence Fluorescent colorants, visual color

The quality control testing of fluorescent colorants by the manufacturers has been based upon attempted simulation of the compounders testing. The colorants are dispersed by either an injection molder, extruder, or a Banbury mixer into HOPE for observation in both mass tone and tint forms. In addition, a common method of display is a side-by-side pressout as performed on a hydraulic press. Carefully trained technicians perform visual observations while those in the fluorescent industry await the development of technology which will allow for adequate computerized color measurement. 

Measurement of Whiteness. The Ciba-Geigy Plastic White Scale is effective in the visual assessment of white effects (79), but the availabihty of this scale is limited. Most evaluations are carried out (ca 1993) by instmmental measurements, utilising the GIF chromaticity coordinates or the Hunter Uniform Color System (see Color). Spectrophotometers and colorimeters designed to measure fluorescent samples must have reversed optics, ie, the sample is illuminated by a polychromatic source and the reflected light passes through the analy2er to the detector. 

Color-order systems, such as the many MunseU collections available from Macbeth, have been described previously. Essential for visual color matching is a color-matching booth. A typical one, such as the Macbeth Spectrahte, may have available a filtered 7500 K incandescent source equivalent to north-sky daylight, 2300 K incandescent illumination as horizon sunlight, a cool-white fluorescent lamp at 4150 K, and an ultraviolet lamp. By using the various illuminants, singly or in combination, the effects of metamerism and fluorescence can readily be demonstrated and measured. Every user should be checked for color vision deficiencies. 

Note In the case of HPTLC plates the detection limit for the visual recognition of the violet = 530 nm) colored chromatogram zones was 20 ng per chromatogram zone. With the exception of the two tetrahydrosteroids the cor-ticosteriods could be detected on TLC plates with fluorescent indicators by reason of fluorescence quenching (Fig. 1 A). Figure 2 illustrates the absorption scans of the separations illustrated in Figures 1A and 1B. 

Note The alternative fast blue salt BB produced the most intensely colored chromatogram zones for visual analysis in daylight, while fluorescence quenching in UV light (A = 254 nm) was greater with fast blue salt B and fast blue salt RR (Figs. 1 and 2). 

A variation on this method, called fluorescent in situ hybridization (FISH), uses fluorescent-labeled DNA and RNA probes for detection and visualization of single cells by microscopy or flow cytometry.7 80 The FISH technique is popular because of its sensitivity and speed of visualization fluorescent dyes can be used to produce probes with different colors for simultaneous detection of several organisms.76,81,82... 

The data can be visualized in several formats. In a gel image, the optical density at each point is related to the fluorescence intensity false color images can be used to improve the dynamic range of visualization. We usually employ a logarithmic compression to help visualize the wide dynamic range of the data the image can be processed to saturate the most intense components, allowing observation of less intense components. 

The fluorescent properties of NHS-fluorescein are similar to FITC. The wavelength of maximal absorbance or excitation for the reagent is 491nm and its emission maximum is 518nm, exhibiting a visual color of green (Sheehan and Hrapchak, 1980). Its molar extinction coefficient at 491 nm in a pH 8.0 buffer environment is Other components in... 

Compounds that fluoresce under ultraviolet light can be visualized in the tissue sections and their locations recorded with color film. Whole-body tissue sections can be used for histochemical localizations for comparison with the autoradiograms. Furthermore, the areas can be removed, extracted, and the extract chromotographed to identify the chemical nature of the radioactivity revealed by the autoradiogram. 

Make it visible the fluorophore label can be visualized directly using fluorescent microscopy. The biotin label (see Sect. 6.2.1) can be detected using streptavidin conjugated with an enzyme the latter must be visualized through an enzyme chromogenic system. Incubate sections with an appropriate enzyme substrate until optimal color develops (see Sect. 2.3). 

Figure 1.31. Fluorescence microscopy pictures to visualize the diffusion of Ox+ to Py+ in zeolite L. The images were taken after (1) 20 min, (2) 60 min, (3) 470 min, and (4) 162 h, respectively. They were obtained by exciting Py+ at 470-490 nm, with the exception of 3b where Ox+ was specifically excited at 545-580 nm. Two crystals of each image are framed. The scale given in 1 corresponds to a length of 1.5 pm (ppy+ = Pox+ — 0.008). (See insert for color representation.)...

Fluorescence detection relies on the visualization of a secondary antibody that has been labeled with a fluorophore such as fluorescein (FITC), Texas Red, Tetramethyl rhodamine (TRITC), or R-phycoerythrin. Although this method of detection has a reduced sensitivity of twofold to fourfold compared to chemiluminescence detection, it presents a tenfold greater linear dynamic range, thus providing better linearity and better quantiflcation within the detection limits. Since secondary antibodies can be labeled with fluor-ophores of distinct colors, multiplexing (simultaneous detection of several antigens) of the same blot is feasible. 

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