Although there can be problems getting phycobiliprotein conjugates into intracellular antigens, we find that allophycocyanin (APC) conjugates are brighter and more photostable than Cy5 conjugates when they can be used. R-PE conjugates may work too, although the are even larger and may bleach faster. We have been working on an interesting approach to multicolor detection in imaging using dyes that have extremely high spectral overlap. One of the solutions is to simultaneously use conjugates of fluorescein and the Alexa Fluor 488 dye targeted to spatially separated targets. By following the time-dependent intensity of these two stains (not excited state lifetime) as they photobleach on a pixel-by-pixel basis and then reconstructing the fast-bleaching (fluorescein) pixels in green and the slow bleaching (Alexa Fluor 488) pixels in red, one can get an excellent representation of the actual targets. We have made pictures and a video of this that will eventually be at our Web site. We have, for instance, separated actin and tubulin staining with this method, despite their overlaps in position. A further benefit is complete registration of the images with no chromatic aberration. One can possibly extend this to “three colors of green” by addition of Oregon Green 488 conjugates, which have an intermediate rate of photobleaching. We are working on other nontraditional means of resolving strongly overlapping but spatially resolved green-fluorescent signals (there are several more possible!). Of course, one could also combine this pair with other dyes to get additional targets. Such techniques should work particularly well for FISH.
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