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and offset separately for each fluorescence signal (background 0, saturation 255) so that each fluorochrome is displayed using a full 0 255 gray-scale range (objective, easy to do, and easy to explain and describe to others) The images are then processed if necessary and merged This is a conventional way to acquire and display multicolor images; however, because each signal is acquired to fill the gray-scale range, this method does not allow you to determine the relative amplitudes of the two signals in the original specimen, only the relative signal strength within a given color channel In a perfectly aligned fluorescence imaging system, a point source in the specimen is perfectly registered, pixel for pixel, by different filter sets at the image plane in the camera However, inadequate color correction of the objective lens or poorly aligned filters can cause misregistration of fluorescence signals during color merging.

This artifact is recognizable by the uniform displacement of signals In images with complex patterns and a mixture of bright and dim signals, this can go undetected, leading you to conclude that signal distribution in a structure is distinct or partially overlapping Therefore, during a color merge operation, in order to obtain good registration, it is important to be able to freely move the color layers with respect to each other, an operation called panning The ability to align through panning requires the presence of coincident reference points (prominent features in the object) in each color layer If multiply stained reference points do not exist, it may be useful to add multifluorescent beads to the specimen, at a dilution giving just a few beads per field of view, prior to mounting with a coverglass.

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For critical applications, there is another solution: Use a single multifluorescence filter cube with a multiple-bandpass dichroic mirror and a barrier filter together with different fluorochrome-specific exciter filters so that the same dichroic mirror is used for all of the fluorescence signals; this configuration is used in confocal microscopes and other wide-field fluorescence microscopes where color alignment is critical More problematic is the case where the microscopist s acquisition and processing methods are uneven Inaccurate interpretations regarding colocalization in merged color images can result from improper gain and offset settings of the camera during acquisition or from extreme histogram stretching during image processing This is depicted in a sketch in Figure 16-2 It is important to follow the procedures outlined in this book;.

lites While it is impractical to discuss specialized labeling techniques and methods of fluorescence quantitation and analysis, it is important to note some of the principal applications for which fluorescence microscopy is applied These include: Determination of the intracellular distribution of macromolecules in formed structures such as membranes, cytoskeletal filaments, and chromatin Fluorochromeconjugated metabolites, ligands, and proteins can be used to label membrane channels and ion channels Target molecules can also be labeled with fluorescent antibodies (immunofluorescence microscopy) or with biotin or epitope tags, or conjugated to fluorescent proteins such as green fluorescent protein (GFP) and other agents Multicolor labeling is possible, whereby several different molecular species are labeled and viewed simultaneously using dyes that fluoresce at different wavelengths Study of intracellular dynamics of macromolecules associated with binding dissociation processes and diffusion (fluorescence recovery after photobleaching, or FRAP).

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FRAP techniques give the halftime for subunit turnover in a structure, binding constants, and diffusion coefficients Study of protein nearest neighbors, interaction states, and reaction mechanisms by fluorescence energy transfer or FRET and by fluorescence correlation microscopy In FRET, two different fluorochromes are employed, and the excitation of the shorter-wavelength fluorochrome results in the fluorescence of the longerwavelength fluorochrome if the two moieties come within a molecular distance of one another Study of dynamics of single tagged molecules or molecular assemblies in vivo using an extremely sensitive technique called total internal reflection fluorescence (TIRF) microscopy Determination of intracellular ion concentrations and changes in the concentrations for several ionic species, including H , Na , K , Cl , Ca2 , and many other metals Ratiometric dyes are used, whose peak fluorescence emission wavelength changes depending on whether the dye is in the free or bound state.

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