Characterization of unexpected blue-shifted single molecule fluorescence of JF646 and JF669 for SMLM

JF-Halotag dyes have become increasingly popular for single-molecule localization microscopy (SMLM) because of their favorable photo-physics, ease of use, and compatibility with live-cell imaging. However under a variety of conditions, the excitation and emission wavelengths from individual emitters can deviate significantly from the commonly accepted characterization, leading to artifacts in two color SMLM data. Here I show results from multiple in vivo and in vitro experiments demonstrating 640nm mediated photo switching of JF669 from a conventional far-red signal to a 561nm excitable red single molecule signal. This result is exciting because it enables UV-free PALM experiments. UV radiation causes cell death, increases auto fluorescence, and induces cell stress, which may affect the behavior of labeled proteins or cellular processes of interest. Additionally, I present evidence of a subpopulation of JF646 molecules that can be excited by 561nm light, but emit red instead of far-red light. This unexpected fluorescence causes single molecule cross talk in the red channel, despite the commonly accepted characterization. This phenomenon is more apparent at low dye concentrations as well as high temperatures, suggesting that a self-interaction between JF646 molecules may bias the well characterized fluorescence, while in isolation JF646 may have significantly blue-shifted excitation and emission spectra. Together, these results show potentially useful photo-physical behavior of JF dyes, but also highlight the importance of careful controls when using synthetic dyes for single molecule localization microscopy.