I’ve been using your Cyanine7.5 amine to label some of my polymers, but I can’t seem to get any signal when I test the fluorescence on the fluorometer. I also just tried the dye alone and couldn’t get any signal. Why might this be? I tried in both water and DMF.
Possible issues may be:
- Wrong settings of excitation and emission wavelengths. For Cyanine7.5, try to set excitation to 780 nm and emission to 810 nm. Slits should be set to ⩽10 nm for this setup. The information on excitation and emission data for other dyes are available in Fluorophore - Reactive group selection chart.
- Fluorometer might not be able to operate in near-infrared. Some fluorometers require the installation of NIR-sensitive photomultiplier tubes (PMT) to operate in NIR. For example, R928 PMT should be installed in the Varian Cary Eclipse fluorometer. Please check if your instrument is compatible with NIR.
- Concentration may be too high, or aggregation can take place. To troubleshoot, try to prepare a dye solution in ethanol having an optical density of 0.1-0.3 at 790 nm. You will need only a tiny amount of the dye.
I am able to get a signal on my instrument when either the dye alone or the dye on a polymer is dissolved in ethanol. However, I cannot get a signal in water which I can somewhat understand is due to aggregation-induced quenching. However, I would have expected a signal to occur for these materials dissolved in DMF, which I cannot see. Why might this be? Is fluorescence also solvent dependent?
- Regarding the signal from non-sulfonated dyes in water - yes, this is the expected behavior to induce quenching due to aggregation. In this case, we advise you to conjugate the dye with something that prevents aggregation (for example, attach the dye to a big protein molecule), and you will be able to see the signal.
- The use of sulfonated dyes can resolve this problem. However, amino-derivatives of sulfonated dyes are zwitterions and have somewhat lower solubility - but should be enough to observe the signal. Or you can use other derivatives such as carboxylic acid or almost any other - they are very well soluble. Non-sulfonated dyes are expected to give a good signal in DMF. It solvates them well. If not, then the concentration is too high.