Seeing in the dark: Vectashield quenches Alexa Fluor 647 fluorescence, but does not hinder dSTORM imaging

It was recently reported that Vectashield, a commercial mounting medium, can be used for direct stochastic optical reconstruction microscopy (dSTORM) of Alexa Fluor 647 (AF647). However, while testing its compatibility with dSTORM imaging of neurofilament light chain (NfL), we noticed an unexpected loss of AF647 fluorescent signal in Vectashield. A quantitative analysis shows that there is an intensity drop to about 15% of the initial AF647 intensity in phosphate-buffered saline (PBS). Interestingly, lost AF647 signal can be partially recovered after washing. On the contrary, for Alexa Fluor 488, we saw an increase in intensity in Vectashield compared to PBS. Our results suggest that AF647 in Vectashield is not compatible with conventional fluorescence microscopy, because the dye gets quenched. Surprisingly, even though we can hardly identify positively labeled cells in Vectashield, we can still perform 3D dSTORM imaging of NfL. However, this is only possible by identifying labeled cells in PBS before adding Vectashield, i.e. before the images turn dark. Otherwise, it is not possible to tell apart background from a labeled cell. Our findings have important consequences not only for dSTORM, but for any type of fluorescence microscopy.