ABSTRACT
Single-molecule localization microscopy (SMLM) enables the visualization of biomolecules at unprecedented resolution and requires control of the fluorescent blinking (ON/OFF) states of fluorophores to detect single-molecule fluorescence without overlapping of the signals. Although SMLM probes based on the intramolecular spirocyclization of Si-xanthene fluorophores have been developed, fluorophores with lower ON/OFF ratios are required for SMLM visualization of high-density structures. Here, we describe a silinane structure that lowers the ON/OFF ratio of Si-xanthene fluorophores. On the basis of Mulliken population analysis, we replaced the dimethylsilane moiety in Si-rhodamine with a silinane moiety to increase the partial charge at the 9-position of the carbon atom in the Si-xanthene ring and to promote the ring-closure reaction. Evaluation of fluorescence properties in a solution and in single-molecule imaging indicated that introducing the silinane sufficiently stabilized the nonfluorescent spirocyclic forms, thus decreasing the fluorescence ON/OFF ratio. This novel substitution was applied to Si-rhodamines with various amine structures and to an Si-fluorescein to expand the color palette. We demonstrated SMLM observation of microtubules in fixed HeLa cells using the developed fluorophores in two color channels. The results demonstrated the feasibility of extending the design strategies of SMLM probes based on Si-xanthenes through modification of the substituents on the Si atom.
