Silicon-rhodamine (SiR) is structurally related to the well-known family of rhodamine fluorophores (e.g. Texas Red and TMR: tetramethylrhodamine), but exhibits a variety of distinctly different and advantageous properties for applications in live cell microscopy experiments (see Figure 1 for structure). SiR is a bright and far-red fluorophore with excitation and emission wavelengths around 650 and 670 nm, a spectral range where very little autofluorescence and phototoxicity occurs. The long wavelength allows a high penetration depth and consequently deeper imaging.
Fig. 1: SiR-based probes for live-cell imaging. (a) SiR derivatives exist in equilibrium between the fluorescent zwitterionic (open) form and the non-fluorescent spiro (closed) form. Binding of the probe to the target of the ligand favors the fluorescent open form, whereas free, unbound SiR probes exist mainly in the closed non-fluorescent form, presumably stabilized by reversible hydrophobic aggregation. (b) Structures of SiR derivatives described in this review. Only the closed spiro form of the fluorophore is shown.
Very importantly, SiRs have been shown to be membrane permeable and therefore allow staining in living cells without tedious transfection protocols.
Another key feature of SiR derivatives is their fluorogenic character. Depending on their environment, they reversibly adopt a non-fluorescent spirolactone form (OFF-state) or a highly fluorescent zwitterionic form (ON-state), respectively (Figure 1). The ON-state is favored by binding to the reaction partner. Therefore, by proper design of SiR-fluorophore based probes, a more than 100-fold increase of fluorescence upon target binding can be achieved. The result is a highly sensitive imaging with low background levels even without removal of excess probe through washing steps.
The use of SiR dyes for live-cell imaging requires the specific labeling of a protein of interest. In general, this is achieved by coupling of SiR to targeting ligands. For example, SiR derivatives that specifically react with self-labeling protein tags such as SNAP-, CLIP or Halo-protein tags have been described . The most popular self-labeling protein tag is SNAP-tag and the corresponding SiR derivative (SiR-SNAP, Figure 1) is commercially available. SNAP-tag is a small protein of 20 kD that specifically and rapidly reacts with benzylguanine (BG) derivatives carrying a fluorophore such as SiR . This permits the specific labeling of SNAP-tag fusion proteins in live cells and even in vivo. Labeling of SNAP-tag fusion proteins with SiR is greatly facilitated by the fluorogenic character of the dye, i.e. its increase in fluorescence intensity upon transfer to SNAP-tag. An example highlighting the potential of SiR-SNAP is the specific and highly efficient labeling of SNAP-tag-expressing cortical neurons in rat brain slices.
The use of self-labeling protein tags such as SNAP-tag is restricted to cells and organisms that are amenable to transfections or genetic manipulations. The cytoskeleton is a structure for which direct fluorescent probes are tremendously useful, as it is involved in a large number of biological processes. To generate suitable probes for live-cell imaging of the cytoskeleton, SiR was conjugated to the microtubule and F-actin ligands docetaxel and desbromo-desmethyl-jasplakinolide, respectively . The resulting SiR-tubulin and SiR-actin probes (Figure 1), available now through the company Spirochrome, are highly fluorogenic and are perfectly suited for live-cell imaging (see Figure 2) as they display very low cytotoxicity. When applied to HeLa cells, SiR-probes did not interfere with the formation of the mitotic cytoskeleton at probe concentrations sufficiently high for imaging: normal metaphase and anaphase spindle morphology as well as normal appearance of the cleavage furrow was observed in presence of 100 nM SiR-actin or SiR-tubulin, respectively. Neither mitotic duration nor proliferation rates were affected at these concentrations. SiR-actin and SiR-tubulin thus allow imaging of the cytoskeleton of normally dividing cells without any obvious toxic effects. Additionally, their red-shifted excitation wavelengths minimize phototoxic effects often seen with probes at shorter wavelengths.
All super-resolution approaches rely on switching between the fluorescent and dark state of the dye. Therefore the attainable resolution is dependent on the spectroscopic properties of the dye. The bright fluorescence, photostability and low phototoxicity render SiR derivatives exceptionally suitable for super-resolution microscopy. Proof-of-principle experiments reveal that SiR derivatives can be used for