Red Light Mediated Photoconversion of Silicon Rhodamines to Oxygen Rhodamines for Single-Molecule Microscopy

The rhodamine motif has been modified in myriad ways to produce probes with specific fluorescent and chemical properties optimal for a variety of fluorescence microscopy experiments. Recently, far-red (>640 nm) emitting silicon rhodamines have become popular in single-molecule localization microscopy (SMLM), since these dyes are membrane-permeable and can be used alongside red (570-640 nm) emitting fluorophores for two-color imaging. While this has expanded multicolor SMLM imaging capabilities, we demonstrate that silicon rhodamines can create previously unreported photoproducts with significantly blueshifted emissions, which appear as bright single-molecule crosstalk in the red emission channel. We show that this fluorescence is caused by the replacement of the central silicon group with oxygen after 640 nm illumination, turning far-red silicon rhodamines (JFX650, JF669, etc.) into their red oxygen rhodamine counterparts (JFX554, JF571, etc.). While this blueshifted population can cause artifacts in two-color SMLM data, we demonstrate up to 16-fold reduction in crosstalk using oxygen scavenging systems. We also leverage this far-red photoconversion to demonstrate UV-free photoactivated localization microscopy (PALM) without the need for additives, and with 5-fold higher efficiency than the Cy5 to Cy3 conversion. Finally, we demonstrate multiplexed pseudo two-color PALM in a single emission channel by separating localizations by their photoactivation wavelengths instead of their emission wavelengths.