Nyomtatóbarát változat

The last decade has seen the exploding evolution of fluorescent dyes that can be used in quantitative microscopic image analysis such as the measurement of fluorescence resonance energy transfer (FRET). Manufacturers claim their dyes to be superior to others in terms of photostability and/or brightness. However, photostability is sometimes a disadvantage (e.g. in acceptor photobleaching FRET), and is often sought to be influenced by scavengers that protect the excited state from damage by free radicals, such as Mowiol. Furthermore, brightness and photostability are both a function of excitation power, which is often overlooked. We have analyzed a set of most often used fluorescent dyes of the visible spectrum: Fluorescein, AlexaFluor488 and Atto488 in the blue excitation, green emission range, Cy3, AlexaFluor546 and 555 and Atto550 in the green excitation, red emission range, and Cy5, AlexaFluor647 and Atto647N in the red excitation, far red emission range. As a novel compound, we have also assayed Atto RM520, a long Stokes-shift dye that can be excited in the blue, but emits in the far red. Each dyes was conjugated to trastuzumab, an antibody binding the ErbB2 protein, an SKBR3 cells expressing ErbB2 at ~800.000 per cell were labeled for microscopy and flow cytometry. Red emitting dyes were found to be far more stable at illumination powers required for equivalent brightness and are thus recommended as donors in acceptor bleaching protocols, or acceptors in donor bleaching protocols. Green emitting dyes were easily photobleached and found to decay with a double exponential, similar to that determined earlier for fluorescein, which makes them only partially ideal for donor photobleaching FRET experiments. The indocarbocyanine Cy5 and AlexaFluor647 far red dyes were the only to photobleach with a single exponential, and were the least stable, which make them ideal for acceptor photobleaching using any of the red dyes as the donor. This photolability is balanced by the smaller contribution of autofluorescence to the signal in this spectral range. The Atto647N proved to be exceptionally stable as advertised, but underwent various photochemical conversions as suggested by its initially rising intensity during continuous exposure. The scavenger Mowiol both quenched and protected from photobleaching most of the tested dyes, but produced a variable adverse effect with Cy5, often accelerating its bleaching rate. The Atto dyes proved to be considerably less bright than their Alexa and indocarbocyanine peers throughout the spectrum. The stability and brightness of these two letter groups was comparable for each spectral window. Overall, the photostability of all examined green, red and far red dyes make them applicable to their practical roles in photobleaching FRET - the less stable greens and far reds to be donors and acceptors in their respective bleaching protocols, and the reds to be both stable acceptors or donors, respectively.