Targeted Photoswitchable Probe for Nanoscopy of Biological Structures (2010)

SNAP-tag: We introduce a photoswitchable O6-benzylguanine derivative and demonstrate its use for super-resolution microscopy of SNAP-tagged proteins based on single fluorophore localization. Stochastic Optical Reconstruction Microscopy (STORM) reveals SNAP-tagged microtubule structures with ∼25 nm resolution. The described probe in combination with the versatile SNAP-tag labeling opens new possibilities for imaging biological structures at the nanoscale. The advent of optical technologies that are capable of imaging structures with a resolution well below the diffraction limit has provided new insights into biology.1 One particularly attractive approach for nanoscopy that is based on the high-precision localization of single emitters is stochastic optical reconstruction microscopy (STORM) and related techniques.2 STORM has been successfully used for the imaging of different biological structures with nanoscale resolution.2a, 2c, 3 It requires the use of photoswitchable or blinking probes. This can be realized with pairs of cyanine dyes,1c, 4–6 single dyes (i.e., as in dSTORM5 and fluorescent proteins, among others)6 or even in a multicolor fashion. These switches can be cycled between “on” and “off” states many times; this allows modulation of the fluorescence emission of individual fluorophores in time such that only an optically resolvable subset of fluorophores is activated at any moment. Nanoscale super resolution is then achieved by precise localization of single emitters across all acquired frames.1a, 1c, 3e The accuracy of STORM imaging relies on the attachment of the photoswitchable probe that is specific to the structure of interest (as well as certain other factors, see the Supporting Information). This has commonly been achieved by chemically labeling antibodies with pairs of cyanine dyes; the antibody then targets the photoswitchable probe to the structure of interest.1c, 3c, 3d However, this approach has a number of problems. First, for each structure of interest, monoclonal antibodies with high specificity and affinity are needed. Second, the random chemical labeling of the antibody with the photoswitchable probes leads to heterogeneous samples. Third, antibodies are large proteins of 150 kD; this results in an artificial increase in size of the imaged structures.4, 5, 7 In principle, these limitations could be addressed by using self-labeling proteins to introduce photoswitchable probes suitable for STORM.8 The SNAP-tag is a small and highly soluble protein of 20 kD, and this makes it an ideal candidate for the construction of fusion proteins for nanoscopy; an example of this is the recent application for the stimulated emission depletion (STED) approach.9 It has been previously demonstrated that SNAP-tag fusion proteins can be labeled with a wide variety of different benzylguanine (BG) derivatives in cells.10 The labeling is highly specific, fast, stoichiometric and quantitative. Furthermore, SNAP-tag labeling can be achieved after fixation of cells; this is a prerequisite for its compatibility with STORM. Here, we apply SNAP-tag in combination with a photoswitchable substrate for the imaging of microtubules with an estimated resolution of ∼25 nm (FWHM, see Figure S12). Specifically, we synthesized a probe that contains Cy3 and Cy5 dye molecules as well as a BG moiety (Scheme 1). The BG permits the targeting of the Cy3-Cy5 photoswitch to SNAP-tag fusion proteins. Photoswitchable probe BG-Cy3-Cy5 for labeling of SNAP-tag fusion proteins of interest (POI). We synthesized BG-Cy3-Cy5 in four steps from commercially available materials and demonstrated that the probe reacts with the SNAP-tag in vitro (Figures S1 and S5). We then attempted to use the probe for nanoscopy of microtubules, which are a copolymer of α- and β-tubulin.11 Towards this end, we expressed β-tubulin as a C-terminal fusion of SNAP-tag (β-tubulin-SNAP) in U2OS cells. To verify the applicability of BG-Cy3-Cy5 for the labeling of proteins in cells, we fixed U2OS cells expressing β-tubulin-SNAP and incubated them with the probe; this incubation was followed by a washing step. As a control, we performed α-tubulin immunostaining of the cells in parallel. Confocal fluorescence imaging demonstrates a highly specific labeling of β-tubulin-SNAP with Cy3-Cy5, as shown by the colocalization of the signals from the immunostaining of α-tubulin and the SNAP-tag labeling (Figure 1). It should also be noted that SNAP-tag remains active and can be labeled after methanol fixation, whereas green fluorescent protein (GFP) is known to lose fluorescence under similar conditions.12 Confocal fluorescence images of cells expressing β-tubulin–SNAP fusion protein. Overlapping of the fluorescence resulting from the chemical labeling and immunostaining indicates the correct localization of β-tubulin–SNAP to the microtubules and demonstrates the specificity of the approach. Cellular α-tubulin was stained using mouse anti-α-tubulin and Alexa 488 labeled anti-mouse IgG antibody (green). Transiently expressed β-tubulin–SNAP fusion protein was labeled with the SNAP-tag substrate BG-Cy3-Cy5 (red). Nuclear DNA staining with Hoechst 33342 was used as a reference (blue). Cells were fixed with methanol prior to staining. The labeled samples were imaged using a custom designed inverted total internal reflection (TIRF) microscope in epi-illumination. The samples were embedded in an imaging buffer containing mercaptoethanol and an oxygen scavenging system prior to imaging.2b Two laser excitation sources were used: a red HeNe laser (633 nm, 2.5 mW) for imaging and a green argon laser (514 nm, attenuated to 200 μW) for activating the photoswitchable tags (Figures S6 and S7). For the STORM image reconstruction, 5000 images were acquired at a 20 Hz frame rate. Reconstructed images after single molecule localization were filtered using an image processing freeware to remove the nonstructured background without any significant loss of information (see the Supporting Information). An example of a representative image analysis sequence is depicted in Figure 2. The improvement in resolution when comparing Figure 1 with 2 B and C is already evident; structures in the order of 40 nm become clearly resolvable (Figures 2, S10, and S11). The isolated spots, which are not part of any structure in the image, are most likely free labeled β-tubulin-SNAP or unreacted BG-Cy3-Cy5. The statistical analysis of the image shows a mean width (2σ) of approximately 36 nm (Figure 2 D). Stochastic Optical Reconstruction Microscopy of microtubules labeled with BG-Cy3-Cy5 by β-tubulin–SNAP. A) The wide field microscopy image of a selected cell (normalized color scale by the mean intensity of all frames); B) the complete field of view after STORM reconstruction; C) zoom into the microtubules highlighted in B)–white inset; D) transversal localization distribution on the microtubule fitted to Gaussian distribution. The localization distribution is averaged over a 200 nm large region along the structure (white box in C)). The Gaussian fit shows a mean width (2 s) of approximately 36 nm, a characteristic feature size well below the diffraction limit. The results presented in Figures 1 and 2 show the applicability of our new photoswitchable probes for the specific labeling of SNAP-tag fusion proteins in cells and the subsequent STORM-based nanoscopy. Microtubules visualized with β-tubulin-SNAP have been resolved well below the diffraction limit. The characteristic dimensions of these structures measured by this method are 40±10 nm in diameter; this value is close to the theoretical value of 30 nm, which is obtained from a microtubule diameter of ∼25 nm, and an estimated distance of ∼2.5 nm from the N terminus of the SNAP-tag to its reactive cysteine residue. It is noteworthy that this measured size of the microtubule is smaller than that measured with STORM based on antibody staining (60 nm).2b An additional feature of the chemical labeling is that the fixed stoichiometry of one label per protein permits an estimation of the number of labeled proteins incorporated into the structure of interest. In summary, we have introduced a targeted photoswitchable probe for the labeling of SNAP-tag fusion proteins in cells and have successfully used them for STORM-based nanoscopy. The small size of the SNAP-tag and the simplicity of its labeling with a large variety of fluorescent probes make it well suited for the imaging of biological structures. Furthermore, it should be straightforward to extend the approach described here to multicolor STORM, or dSTORM,5 using different, “orthogonal” self-labeling tags, such as the CLIP-tag.10a We therefore believe that the approach described will significantly broaden the possibilities of performing nanoscopy of biological structures. The authors acknowledge the funding support of the Swiss National Science Foundation (Synergia project CRSII3 125463; K-23K1-116242/1; 315200-116729). G.L. acknowledges the Federation of European Biochemical Societies for a long-term fellowship. The authors thank Francois Aguet and Pierre Gönczy for interesting and helpful discussions. Detailed facts of importance to specialist readers are published as ”Supporting Information”. 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