Fluorescence microscopy, enzyme

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). 

Brennan et al. used a method to detect the reaction of acetylcholineesterase with acetylcholine [46]. The method was based on the use of a monolayer, consisting of fatty acids having Ci6 chain lengths, which were covalently attached to quartz wafers and which contained a small amount of nitrobenzoxadiazole dipalmitoyl phosphatidylethanolamine (NBD-PE) (partitioned from water into the membrane). The enzyme substrate reaction produced a decrease in fluorescence intensity from the monolayer, and the detection system was sensitive to the changes in bulk concentration of as small as 0.1 pM, with a limit of detection of 2 pM of acetylcholine. The mechanism of transduction of the enzymatic reaction was investigated using spectrofluorimetric methods and fluorescence microscopy. 

The technique of immunohistochemistry is very similar to fluorescence microscopy. This technique differs only in the method of detection or localization of the antibody and can be performed with a conventional light microscope. As with the ELISA and Western blot, the antibody used in this experiment is covalently conjugated to an enzyme, such as horseradish peroxidase. This enzyme is then incubated with a substrate that is converted to an insoluble colored product that will precipitate or deposit at the site of enzyme activity. The distribution and location of the colored product is readily detected with an ordinary light microscope. 

The second step required in the application of avidin-biotin technology is to prepare an appropriate avidin-associated probe or probes for the desired application (for general reviews, see refs. 1 and 2). For example, a fluorescent form of avidin can be used for fluorescence microscopy, fluorescence-activated cell sorting, and in some cases, for immunoassay. Likewise, an avidin-enzyme conjugate can be used for immunoblotdng, immunoassay, light microscopy, and in some cases, electron microscopy. An immobilized form of avidin can be used for isoladon purposes (see Note 1). 

The use of aminophenyl fluorescein [28] as the organic compound allows the selective localization of the secondary oxidation reaction with confocal fluorescence microscopy (Fig. 25.3a) [29]. In a similar way as described for a-chymotrypsin, the enzyme was immobilized in an agarose matrix and fluorescence intensity time traces were recorded at a position where an enzyme was found. Time traces with exceptional signal-to-noise ratio were obtained (Fig. 25.3b) and a histogram of time-averaged single enzyme activities was constructed (Fig. 25.3c) which allowed the determination of the average activity of the analyzed enzymes. 

---