Confocal affinity

FIG. 3. Confocal images showing the location of the SR in live myocytes within an intact, small diameter (< 250 nm passive diameter), pressurized (70 mmHg) artery from the rat mesenteric artery arcade. The artery was loaded with Fluo-4 as the membrane-permeant acetoxymethyl ester. Some of this high-affinity, Ca2+ indicator dye is often sequestered in the SR (cf. Goldman et al 1990). The SR can then be readily visualized, especially when [Ca2+]CYx is low (as in the panels at 0 and 6.8 s), because the intra-SR dye is saturated with Ca2+, and fluoresces brightly. This artery was treated with 1.0 fim phenylephrine (PE), which caused the [Ca2+]CYT level to oscillate asynchronously in the cells seen in the centre of the panel. The cell outlines are clearly visible when [Ca2+]CYT tiscs, as in the panels at 3.4 and 10.2 s. Note that nearly all of the SR (the very bright areas, especially in the 0 and 3.4 s panels) lies parallel to, and immediately beneath the PL (from Miriel at al 1999, with permission). 

Schu(ller A, Bonfante P, Schnepf E, Mollenhauer D, Kluge M. Characterisation of the Geosiphon pyriforme symbiosome by affinity techniques confocal laser scanning (CLSM) and electron microscopy. Protoplasma 1996 190 53-67. 

Confocal laser scanning fluorescence microscopy was used to study the exposure of the avidin-specific binding sites in the Av-GEB platform by the immobilization of a small and flexible biotinylated fluorescein molecule as a fluorescence marker. Fluorescence microscopy thus confirms that Av-GEB platform exposes active binding sites for biotin, acting as affinity matrix (Fig. 21.2B). After use, the electrode surface can be renewed by a simple polishing procedure for further uses, highlighting a clear advantage of this new material with respect to surface-modified approaches such as classical biosensors and other common... 

More recently, confocal fluorimetry itself has been impressively extended. In particular, the implementation of multi-photon excitation opened the potential to excite different fluorescent labels by a single laser line [47]. This considerably simplified the optical setup of confocal instruments. For example, Heinze et al. [48] described a setup for two-photon excitation confocal fluorimetry where three molecular species were quantified simultaneously using a single laser. When included in screening systems, these spectroscopic advancements enable the quantification of enzymatic reaction rates on several substrates in parallel or, when applied for peptide or protein ligands, the simultaneous measurement of binding affinities on different target receptors. In this way, biopharmaceuticals can be selected on the basis of their specificity and selectivity. As a consequence, undesired side activities can be controlled very early in the hit identification process. 

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