New Fluorescence Probes for Live-Cell Imaging

Visualizing the Cytoskeleton with SiR-based Probes

  • New Fluorescent Probes for Live-Cell Imaging - Visualizing the Cytoskeleton with SiR-based ProbesNew Fluorescent Probes for Live-Cell Imaging - Visualizing the Cytoskeleton with SiR-based Probes
  • New Fluorescent Probes for Live-Cell Imaging - Visualizing the Cytoskeleton with SiR-based Probes
  • Fig. 1: SiR-based probes for bioimaging. (a) SiR derivatives exist in an equilibrium between the fluorescent zwitterionic (open) form and the non-fluorescent spirolactone (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. For reasons of clarity, only the closed spirolactone form of the fluorophore is drawn.
  • Fig. 2: Live-cell microscopy with SiR-probes. (a) Labeling of the microtubule- and centrosome-binding protein CEP41. Cep41 was expressed as SNAP fusion protein and labeled with SiR-SNAP. (b) Structured illumination microscopy (SIM) images of microtubules in living primary human fibroblasts. (c) SIM images of actin fibers in primary human fibroblasts labeled with SiR-actin.
  • Fig. 3: Live-cell STED microscopy. (a) Images of microtubules stained with SiR-tubulin in human primary dermal fibroblasts. The diameter of the microtubules measured along the white dotted line is around 40 nm. (b) A representative STED image of stained microtubules in the centrosome after Richardson-Lucy deconvolution; the observed ring is a projection of the centriole along its longitudinal axis. The measured polar angle in-between neighboring maxima of fluorescence intensity along the periphery of the centriole was measured to be around 40°. The diameter of the centriole was measured to be around 176 nm. (c) STED image showing axons of rat primary hippocampal neurons stained with SiR-actin. Scale bar, 1 µm. (d) Measured intensity signal profile of SiR-actin stripes fitted to multiple Gaussian distributions and estimation of the interval between neighboring peaks. The measured periodicity of the structure of around 180 nm is within the experimental error of the measured periodicity in fixed samples.

We review here a new generation of far-red fluorescent probes for live-cell imaging, which are based on a novel cell-permeable silicon rhodamine (SiR) dye. These probes combine a variety of desirable features, such as excellent selectivity, fluorogenicity, high brightness and low cytotoxicity, rendering them ideal probes for conventional and state-of-the-art super-resolution microscopy. Application of these new probes in combination with STED microscopy revealed for the first time the nine-fold symmetry of the centrosome and confirmed the spatial organization of actin in the axon of cultured neurons in living cells.

New Fluorescent Probes for Challenging Live Cell Imaging

Live-cell fluorescence microscopy is ideally suited to provide insights into the dynamics of biological processes such as cytokinesis, motility and organelle organization. Furthermore, live-cell imaging of cellular structures avoids the occurrence of fixation artifacts that can occur when working with fixed specimens [1]. While there are thus strong arguments to favor imaging with live cells over fixed cells, performing such experiments can be challenging. One of the difficulties is that most fluorescent probes that work well on fixed samples are not applicable to living specimen. Consequently, there is a generally acknowledged need for new fluorescent probes for live-cell imaging. What properties does a fluorescent probe for live cell imaging need to have? The ideal fluorescent probe for live-cell imaging is non-toxic, permeable, has emission and excitation wavelengths in the far-red, labels the biomolecule of interest with exquisite specificity, does not require genetic manipulation and is fluorogenic (i.e. only becomes fluorescent when binding to its target). Up to now, few probes that fulfill this rather extensive wish list have been described. In particular, far-red synthetic fluorophores, such as Cy5 and Alexa647N tend to be membrane impermeable and/or display unspecific binding to cellular components.

Therefore, in order to overcome these shortcomings, a new class of far-red fluorescent probes based on carboxylated silicon-rhodamine (SiR; fig.

1) was developed recently [2]. SiR is a bright and far-red fluorophore with excitation and emission wavelengths around 650 and 670 nm, respectively, which can be readily derivatized with specific ligands that target the fluorophore to the structure of interest (fig. 1). A key feature of such SiR derivatives is that they exist in equilibrium between a non-fluorescent spirolactone form and a highly fluorescent zwitterionic form, respectively. Reversible aggregation of SiR derivatives and/or their unspecific binding to hydrophobic surfaces favors the non-fluorescent state whereas binding to their target and concomitant deaggregation switches the probes into the highly fluorescent state. Depending on the structure of the SiR-probe, the resulting increase in fluorescence upon target binding can be more than 100-fold, thereby exhibiting an excellent signal-to-noise ratio and even permitting imaging of samples without removal of excess probe through washing steps. The latter point is of particular importance when imaging in vivo or in tissues, as washout of excess probe in such cases is usually very difficult.

Live-Cell Microscopy with SiR-Probes

One attractive application of SiR-derivatives is their use in conjunction with self-labeling protein tags such as SNAP-, CLIP- or HALO-tag [3]. Such tags permit a specific coupling of synthetic fluorescent probes to the corresponding fusion protein. However, prior to the introduction of SiR, far-red fluorophores such as Cy5 and Alexa647N could not be efficiently coupled to these tags in living cells. The SNAP-tag substrate SiR-SNAP (fig. 1) permitted a specific labeling of SNAP fusion proteins in live cells (fig. 2a) [2]. The fluorogenic character of the probe even enabled the specific labeling and imaging of SNAP-expressing cortical neurons in rat brain sections without any significant background signal.

The use of self-labeling protein tags such as SNAP-tag requires the expression of a fusion protein in the organism of interest. A direct labeling of endogenous proteins would remove such technical hurdles and avoid artifacts arising from (over-) expression of fusion protein, opening up numerous experimental possibilities. The cytoskeleton is a structure for which direct fluorescent probes would be 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 [4]. The resulting SiR-tubulin and SiR-actin probes (fig. 1) showed excellent fluorogenicity in in vitro binding assays. SiR-tubulin increases its fluorescence intensity more than 10-fold when binding to microtubules in vitro whereas SiR-actin displays a more than 100-fold increase in fluorescence. Even more importantly, SiR-tubulin and SiR-actin are perfectly suited for live-cell imaging, as demonstrated for human primary dermal fibroblasts (fig. 2b, c). Similar results were obtained with numerous other cell lines. Furthermore, SiR-actin was used to stain the actin cytoskeleton of intact erythrocytes in whole blood samples from humans [4]. The success of the latter experiment is based on the following three properties of SiR-probes: First, their excitation and emission is in the far-red spectral region and therefore not interfering with the absorbance of hemoglobin. Second, the cell-permeability of the SiR-based probes allows their direct introduction even into cells that are difficult to transfect, such as erythrocytes. Third, the fluorogenic character of the probes enables simple, direct and straightforward detection of target-bound probes without the requirement of subsequent washing steps.

Another important feature of both SiR-actin and SiR-tubulin is their low cytotoxicity. At the concentrations utilized for long-term imaging, neither SiR-tubulin nor SiR-actin exhibited any significant cytotoxicity towards primary human fibroblasts over a period of 24 hours. 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. Furthermore, neither mitotic duration nor proliferation rates were affected at these concentrations. Overall, these data demonstrate that SiR-actin and SiR-tubulin allow imaging of the cytoskeleton of normally dividing cells without any obvious toxic effects.

Live-Cell STED Microscopy

The high photostability of SiR derivatives makes them also well suited for the recently introduced live-cell super-resolution microscopy technique by stimulated emission depletion (STED) microscopy [5]. Staining of living human fibroblasts with SiR-tubulin and subsequent STED imaging revealed peripheral microtubules and the microtubules of the centrosome, respectively. The measured microtubule diameter was around 40 nm (fig. 3a), which represents the highest resolution achieved so far for imaging microtubules in living cells. It is instructive to compare the resolution achieved in live-cell STED microscopy of microtubules labeled either with SiR-tubulin or with SiR-SNAP through labeling of the microtubule-binding protein CEP41. In the latter case, a microtubule diameter of around ­­­80  nm was measured [2]. The higher resolution achieved in the experiments with SiR-tubulin demonstrates impressively that small-molecule probes directly targeting the structure of interest can improve the resolution dramatically. This observation is further corroborated by live-cell STED microscopy of human centrosomes. 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 of 176 nm in diameter (fig. 3b). Furthermore, the STED data showed a pronounced modulation in brightness along the perimeter of the ring (fig. 3b). The measured polar angle ϕ between two neighboring maxima equaled around 40°, which is perfectly consistent with the known 9-fold symmetry of the centriole. Therefore and for the first time, the new SiR-tubulin probe allowed the direct visualization of the centriole's architecture in living cells.

SiR-actin is also well suited for live-cell STED microscopy. For example, SiR-actin-labeling and STED microscopy was used to image evenly spaced, ring-shaped structures based on actin at the rim of axons in live primary rat hippocampal neuron cells (fig. 3c). By this technique, a periodicity of about 180 nm could be determined within these structures (fig. 3d). The same value for the periodicity of this regular spatial arrangement of actin in axons had been first observed on fixed samples and has been described in a seminal paper of X. Zhuang's [6] group. The identity of the results obtained from these two quite different experiments confirms that the proposed structure is indeed accurate. In contrast, attempts to reveal this periodic actin structures with the popular actin marker LifeAct [7] failed. The superiority of SiR-actin over LifeAct in these measurements is most likely due to the higher selectivity of SiR-actin for f-actin over g-actin.

Conclusion

The generation of SiR-based probes for other cellular structures and proteins should be feasible as long as an appropriate ligand (i.e. permeable and amenable to derivatization) is available. The recent description of a SiR-based probe for PARPs by the Weissleder group confirms this assumption [8]. In conclusion, SiR derivatives represent a superior new generation of sophisticated far-red fluorescent probes that are ideally suited for live-cell (super-resolution) microscopy. In particular the recently introduced SiR-actin and SiR-tubulin should become popular probes for the imaging of the cytoskeleton of living cells. Moreover, we expect that the unique combination of several attractive features of SiR will stimulate the creation of an entire family of powerful new probes for bioimaging.

Acknowledgment
The authors would like to thank Grazvydas Lukinavicius for preparing figures 2 and 3 and Luc Reymond for discussions.

References
[1] Hormeno S. et al.: Biophys J 97, 1022-1030 (2009)
[2] Lukinavicius G. et al.: Nat Chem 5, 132-139 (2013)
[3] Hinner M.J. & Johnsson, K.: Curr Opin Biotech 21, 766-776 (2010)
[4] Lukinavicius G. et al.: Nat Methods 11, 731-733 (2014)
[5] Hell S.W. & Wichmann J.: Opt Lett 19, 780-782 (1994)
[6] Xu K. et al.: Science 339, 452-456 (2013)
[7] Riedl J. et al.: Nat Methods 5, 605-607 (2008)
[8] Kim E. et al.: Chemical Communications 50, 4504-4507 (2014)

Authors
Prof. Kai Johnsson
(corresponding author via email request button below)
Ecole Polytechnique Fédérale de Lausanne (EPFL)
Institute of Chemical Sciences and Engineering (ISIC)
Institute of Bioengineering
NCCR in Chemical Biology
Lausanne, Switzerland

Dr. Stefan Pitsch
Spirochrome SA
Stein-am-Rhein, Switzerland 

Contact

EPFL Ecole Polytechnique Federale de Lausanne

1015 Lausanne
Switzerland

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