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314      IMAGE PROCESSING FOR SCIENTIFIC PUBLICATION

                                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 nec-
                                essary 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 speci-
                                men, 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 fil-
                                ters 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, dur-
                                ing 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 pan-
                                ning. 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 refer-
                                ence 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 cover-
                                glass. 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 dif-
                                ferent 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 acquisi-
                                tion 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;



                                              I                          I
                                            b

                                            a




                                                    Colocalized               Not colocalized

                                              I                          I

                                            a                         b
                                Figure 16-2
                                Colocalization of two fluorescent signals. The signals represented by the solid and dotted
                                patterns are interpreted to be partially colocalized or have distinct distributions depending on
                                the offset applied to the image. Bottom left: overlaid solid and dotted images using the offset
                                at position a. Bottom right: overlaid solid and dotted images using the higher offset setting at
                                position b.
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