Silinanyl Rhodamines and Silinanyl Fluoresceins for Super-Resolution Microscopy

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.

Small-molecule fluorogenic probes for mitochondrial nanoscale imaging

“…For SMLM imaging, xanthene-based ''photo click'' chemistry has been widely explored. 194 In 2020, Morozumi et al developed a fluorogenic probe by incorporating a benzylchloropyrimidine unit, which is a known substrate for SNAP-tag, in the fluorogenic probe CP550. 195 The CP550-labeled BnClPy (a substrate for SNAP-tag) could be used to image live cells expressing SNAP-tag proteins in mitochondria.…”

Photochemical Mechanisms of Fluorophores Employed in Single‐Molecule Localization Microscopy

“… [97] This means spontaneous blinking is possible, even when the intramolecular nucleophile remains a carboxy group. [98] Silicon‐rhodamine (SiR‐carboxyl, Figure 11 b) was used in super‐resolution imaging (STORM/GSDIM). [99] Iwanaga and co‐workers reported that changing the functionalization on the Si atom from dimethyl to a cyclic silanyl further increases the electrophilicity of the xanthene core.…”

“… [99] Iwanaga and co‐workers reported that changing the functionalization on the Si atom from dimethyl to a cyclic silanyl further increases the electrophilicity of the xanthene core. [98] This increases the likelihood of spirocyclization and results in a lower on/off ratio. They also further modified the rhodamine scaffold by changing the amino auxochrome groups to give a 7‐azabicyclo[2.2.1]heptane substituted analogue, SiliR14 (Figure 11 b), which showed superior photostability and spontaneous blinking properties.…”

Photochemical Mechanisms of Fluorophores Employed in Single‐Molecule Localization Microscopy

“…Silicon‐bridged rhodamine analogues have lower p K cycl values than the corresponding O‐bridged rhodamines, as the LUMO is stabilized by the silicon atom [97] . This means spontaneous blinking is possible, even when the intramolecular nucleophile remains a carboxy group [98] . Silicon‐rhodamine (SiR‐carboxyl, Figure 11b) was used in super‐resolution imaging (STORM/GSDIM) [99] .…”

“…Silanyl-substituted fluorescein SiliF was also synthesized, which allowed the authors to perform dual-color SMLM imaging. [98] Urano and co-workers used a quantum-chemical calculation and rational design approach to design and synthesize a carbon-bridged rhodamine derivative HMCR550 that spontaneously blinks. [100] This method allows the blinking properties of a candidate fluorophore to be predicted, without the need to synthesize a variety of potential structures and obtain experimental measurements.…”