The Alexa Fluor 488 detection channel (see Figures 1(a) and 1(d)) photomultiplier slit has been set to a 30-nanometer bandwidth (ranging from 500 to 530 nanometers) that encompasses the primary probe emission peak, but does not capture a significant amount of fluorescence from C圓 emission. By sequentially scanning the specimen with the individual lasers and detecting fluorescence in each channel to coincide with laser illumination (Figure 1(d) and 1(e) discussed in more detail below), spectral bleed-through is minimized (compare Figure 1(c) to Figure 1(f)) to produce a more accurate merged image of fluorophore distribution. This artifact can be easily confused with co-localization of the fluorophores. Note the Alexa Fluor 488 fluorescence bleed-through apparent in the C圓 detection channel (Figure 1(b)), which is manifested by yellow overlap regions in the final merged image (Figure 1(c)). The pair of images illustrated in Figures 1(a) and 1(b) were obtained by simultaneous lateral scanning of the specimen using a 488 nm laser (Figure 1(a)) and a 543 nm laser (Figure 1(b)). These fluorophores exhibit absorption and emission spectra similar to fluorescein and rhodamine, although with slightly different peak wavelengths and somewhat narrower bandwidths. Spectral bleed-through in a thin section of mouse intestine labeled with Alexa Fluor 488 and C圓 (a cyanine dye), and imaged with a laser scanning confocal microscope having adjustable photomultiplier detector slits is illustrated in Figure 1. Furthermore, fluorescein emission will be detected in the photomultiplier channel or widefield filter set reserved for rhodamine. Thus, excitation of fluorescein using a 488 nm laser will also produce excitation of rhodamine, although to a lesser degree. This effect is due to the fact that these dyes have very broad absorption and emission spectra that exhibit a significant degree of overlap. For example, when double labeling with the traditional green and red probes, fluorescein and rhodamine, bleed-through can only be reduced by using optimized fluorescence filter sets (and/or photomultiplier detector slit widths), but is never completely eliminated. Imaging specimens having two or more fluorescent labels (or plant tissue sections exhibiting a high degree of autofluorescence) is often complicated by the bleed-through or crossover of fluorescence emission, unless the spectral profiles of the fluorophores are very well separated.
Bleed-through artifacts often complicate the interpretation of experimental results, particularly if subcellular co-localization of fluorophores is under investigation or quantitative measurements are necessary, such as in resonance energy transfer ( FRET) and photobleaching ( FRAP) studies. The phenomenon is usually manifested by the emission of one fluorophore being detected in the photomultiplier channel or through the filter combination reserved for a second fluorophore.
Bleed-through (often termed crossover or crosstalk) of fluorescence emission, due to the very broad bandwidths and asymmetrical spectral profiles exhibited by many of the common fluorophores, is a fundamental problem that must be addressed in both widefield and laser scanning confocal fluorescence microscopy.
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