Paper

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

Dalluge, Joseph J., Puchner, Elias M., Noiraeux, Vincent, Ritz, Jacob M., Khakimzhan, Aset

2024

Overview

The rhodamine motif has been modified in myriad ways to produce probes with specific fluorescent and chemical properties optimal for a variety of microscopy experiments. Recently, far-red emitting silicon rhodamines have become popular in single-molecule localization microscopy (SMLM), since these dyes are membrane-permeable and can be used alongside red fluorophores for two-color imaging. While this has expanded multi-color 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 photo-activation wavelengths instead of their emission wavelengths.

• Publisher: Cold Spring Harbor Laboratory
• Subject: Biophysics