What molecules in media present the biggest problems with fluorescence imaging?

Answer

Many of the chemicals in media fluoresce when the right wavelength light is present. Fluorescence microscopy is often difficult since getting the correct signal:noise is dependent on the components that make up the media. Often incubations in PBS are used to reduce auto-fluorescence but there are problems with that since the nutritional content of PBS is basically nonexistent. This problem sets limits to incubation times one can use, if cell health is important. Many components of media are cyclic and conjugated and contribute to noise during fluorescence microscopy because those molecules get excited by the wavelengths used during fluorescent microscopy. Probably phenol red and flavin molecules contribute the most to background fluorescence. This is one reason many people use phenol red-free medium during these types of experiments. The problem though is that when using phenol red-free DMEM there is only a small reduction in background fluorescence compared to PBS.