A Bright Dye for Live-Cell STED Microscopy

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.

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 [1]. 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 [3]. 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 [2]. 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 GSDIM/dStorm (ground state depletion followed by individual molecule return/direct stochastical optical reconstruction microscopy) and SIM (structured illumination microscopy). But particularly the combination of STED microscopy and SiR-based probes is an extremely powerful approach for super-resolution live-cell microscopy. For example, the use of STED microscopy and SiR-labeling of SNAP-tag fusion proteins has been exploited to determine the localization of centrosomal proteins in living cells with unprecedented resolution. However, the most impressive results have been achieved using live-cell STED microscopy and SiR-actin and SiR-tubulin.

SiR-actin-labeling and STED microscopy confirmed the ring-shaped actin-structures at the rim of axons in live primary rat hippocampal neurons (Figure 3a, b). This regular spatial arrangement of actin in axons had been first observed by super-resolution microscopy on fixed samples by X. Zhuang’s group [4]. Importantly, the same periodicity of actin in axons was now observed also in live samples. The characterization of biological structures in live cells is of great importance as it avoids fixation artifacts.

Similar observations were made with SiR-tubulin. The peripheral microtubules and microtubules of the centrosome could be revealed in SiR-tubulin stained living human fibroblasts by STED microscopy [2] with the highest resolution achieved so far for imaging microtubules in living cells: The measured microtubule diameter in this study was around 40 nm (Figure 3c). This also demonstrates that probes directly targeting microtubules or other structures of interest can significantly improve resolution: Live-cell STED microscopy of microtubules labeled with SiR-SNAP and microtubule-binding proteins yielded a microtubule diameter of around 80 nm, i.e. a two-fold lower resolution than what was obtained with SiR-tubulin. Furthermore, the combination of STED microscopy and SiR-tubulin permitted for the first time the direct visualization of the centriole’s architecture in living cells. The key component of the centrosome is the centriole, a cylindrical structure composed of nine triplets of microtubules. Imaging the centrosome in human fibroblasts stained with SiR-tubulin revealed rings that showed a clear modulation in brightness along their perimeter (Figure 3d). The measured polar angle between neighboring maxima equaled around 40°, which is perfectly consistent with the known 9-fold symmetry of the centriole. These and other live-cell STED microscopy experiments with iR-based probes demonstrate the enormous potential of the approach.

In summary, silicon-rhodamine derivatives come very close to be optimal probes for live cell super-resolution microscopy as they

• are bright and photostable;
• are excited in the far-red;
• are fluorogenic;
• show little toxicity; and
• are cell permeable and biocompatible.

STED microscopy with SiR dyes delivers bright images at very high resolution with moderate bleaching. In one sentence: SiR-fluorophore based probes and STED microscopy are the perfect match for live cell imaging below the diffraction limit.

SiR-SNAP is available from New England Biolabs Inc.: www.neb.com

SiR-Actin and SiR-Tubulin are available from Spirochrome Ltd.: www.spirochrome.com

The authors are grateful to Grazvydas Lukinavicius, EPFL Lausanne, for providing the images.

1. Lukinavičius G et al.: A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5: 132–39 (2013).
2. Lukinavicius G et al.: Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat. Methods 11: 731–33 (2014).
3. Keppler A et al.: A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nature Biotechnology 21: 86–89 (2003).
4. Xu K, Zhong G, and Zhuang X: Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons. Science 339: 452–56 (2013).