The dramatic advances in modern fluorophore technology are exemplified by the Alexa Fluor dyes introduced by Molecular Probes (Alexa Fluor is a registered trademark of Molecular Probes). These sulfonated rhodamine derivatives exhibit higher quantum yields for more intense fluorescence emission than spectrally similar probes, and have several additional improved features, including enhanced photostability, absorption spectra matched to common laser lines, pH insensitivity, and a high degree of water solubility. In fact, the resistance to photobleaching of Alexa Fluor dyes is so dramatic that even when subjected to irradiation by high-intensity laser sources, fluorescence intensity remains stable for relatively long periods of time in the absence of antifade reagents. This feature enables the water soluble Alexa Fluor probes to be readily utilized for both live-cell and tissue section investigations, as well as in traditional fixed preparations.

Rat thoracic aorta cells in culture (illustrated above) were immunofluorescently labeled with mouse anti-alpha-tubulin primary antibodies followed by goat anti-mouse secondary antibodies conjugated to Alexa Fluor 568. In addition, the specimen was stained with Alexa Fluor 350 conjugated to phalloidin and SYTOX Green, targeting the filamentous actin network and nuclei, respectively. Images were recorded in grayscale with a QImaging Retiga Fast-EXi camera system coupled to an Olympus BX-51 microscope equipped with bandpass emission fluorescence filter optical blocks provided by Omega Optical. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.