Photostability is a key characteristic of Alexa Fluor® dye conjugates, allowing more time for image capture. In addition to their superior brightness and the wide selection of dyes across the spectrum, you'll get photostability you can rely on with Alexa Fluor® dyes and dye conjugates.
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The photostability of the Alexa Fluor ® dye conjugates is particularly apparent when compared to traditional fluorophores such as fluorescein (Figures 1 and 2).
Figure 1. Comparison of the photobleaching rates of the Alexa Fluor® 488 and Alexa Fluor® 546 dyes and the well-known fluorescein and Cy3 fluorophores. The cytoskeleton of bovine pulmonary artery endothelial cells (BPAEC) was labeled with (top series) Alexa Fluor® 488 phalloidin and mouse monoclonal anti–a-tubulin antibody in combination with Alexa Fluor® 546 goat anti–mouse IgG antibody or (bottom series) fluorescein phalloidin and the anti–a-tubulin antibody in combination with a commercially available Cy3 goat anti–mouse IgG antibody. The pseudocolored images were taken at 30-second intervals (0, 30, 90, and 210 seconds of exposure). The images were acquired with bandpass filter sets appropriate for fluorescein and rhodamine.
Figure 2. Reduced photobleaching, sustained signal. Bovine pulmonary artery endothelial cells were labeled with fluorescein phalloidin (A) or Alexa Fluor® 488 phalloidin (B) which label filamentous actin. The cells were placed under constant illumination on the microscope. Images were acquired at one-second intervals for 30 seconds. Under these illumination conditions, fluorescein images are photobleached to about 20% of its initial value, while fluorescence of Alexa Fluor® 488 phalloidin stayed at the initial value.
Outperforming so-called “Premium” Fluorophores
We have also shown that Alexa Fluor® 488 conjugates outperform many recently introduced “premium” fluorophores (Figure 3). Although several conjugates tested showed good photostability, none outperformed the Alexa Fluor® 488 dye. Several competing conjugates have significantly lower signal intensity, even under optimal conditions.
|Figure 3. Photobleaching analysis results. Mitochondria in bovine pulmonary artery endothelial cells were detected using an anti-OxPhos Complex V inhibitor protein antibody and secondary antibody conjugate of the dye indicated in the graph. Samples were continuously illuminated and data collected every ~250 milliseconds. The working concentrations are noted in the legend.|
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