Live cell imaging of DNA with SiR-DNA for STED & SIM

In their most recent publication “SiR-Hoechst, a far-red DNA stain for live-cell nanoscopy” in Nature Communications, Lukinavičius et al. (1) define ideal characteristics of a nuclear stain for live cell imaging approaches. The stain should…

• show high selectivity
• minimal toxicity
• be fluorogenic for wash-free imaging
• be applicable in different cell types and tissues
• be excited by far-red light
• be suitable for super-resolution microscopy

None of the nuclear stains on the market meets all these crucial requirements leading thus to the need for a new tool for live cell staining experiments of the nucleus.

SiR-DNA – A new nuclear stain which meets all requirements for live cell imaging

The authors describe the development of the new stain SiR-DNA by Spirochrome AG, which is available through tebu-bio on the European market.

The stain carries the bright and photostable fluorophore silicon rhodamine (SiR), a far-red dye which is compatible with most microscopic methods. It can be used with standard Cy5 settings.

The combination of all these properties sets SiR-based probes apart from other fluorescent probes. The SiR-technology has been successfully used to conduct actin and tubulin staining in living cells (2, 3). SiR-Actin and SiR-Tubulin have been a real break-through in cell biology, as they open the door to directly imaging the major components of the cytoskeleton in living cells without the need to transfect cells with vectors carrying the information for florescently labelled proteins.

SiR has been coupled to the DNA minor groove binder bisbenzimide (Hoechst) to form SiR-DNA which meets all requirements listed above.

Through its fluorogenic Hoechst portion it is highly specific, SiR-DNA does not impair cell proliferation within a 24 h measurement interval when used in concentration needed for DNA staining, SiR is a far red probe (Abs: 652 nm; Em: 674 nm), and it is compatible with superresolution microscopy.

SiR-DNA has been shown to work with a number of mammalian cell types as well as in organism imaging of Drosophila.

Typical results with SiR-DNA are shown in Fig. 1 and 2. Note that Fig. 1 also shows the molecular structure of SiR-DNA.

Mitosis in Hela cells can be also followed in this video:

Movie of Live HeLa cells stained with SiR-DNA. Data collected by confocal imaging. Courtesy of Daniel Gerlich and Claudia Blaukopf, Institute of Molecular Biotechnology, Vienna

In Fig 3 and the following video you can see a spheroid formed by MCF10A cells and stained with SiR-DNA.

3D reconstruction from a z-stack of MCF10A cells spheroid stained with SiR-DNA. Courtesy of Christian Conrad and Katharina Jechow, Heidelberg.

Interested in the new SiR-DNA stain? Contact us by leaving a comment below!

References:

1. Gražvydas Lukinavičius, Claudia Blaukopf, Elias Pershagen, Alberto Schena, Luc Reymond, Emmanuel Derivery, Marcos Gonzalez-Gaitan, Elisa D’Este, Stefan W Hell, Daniel Wolfram Gerlich and Kai Johnsson, SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy. Nat. Commun. 6:8497 doi: 10.1038/ncomms9497 (2015)
2. Gražvydas Lukinavičius, Keitaro Umezawa, Nicolas Olivier, Alf Honigmann, Guoying Yang, Tilman Plass, Veronika Mueller, Luc Reymond, Ivan R. Corrêa Jr, Zhen-Ge Luo, Carsten Schultz, Edward A. Lemke, Paul Heppenstall, Christian Eggeling, Suliana Manley and Kai Johnsson, A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins, Nature Chemistry, 5, 132–139 (2013)
3. Gražvydas Lukinavičius, Luc Reymond, Elisa D’Este, Anastasiya Masharina, Fabian Göttfert, Haisen Ta, Angelika Güther, Mathias Fournier, Stefano Rizzo, Herbert Waldmann, Claudia Blaukopf, Christoph Sommer, Daniel W Gerlich, Hans-Dieter Arndt, Stefan W Hell and Kai Johnsson, Fluorogenic probes for live-cell imaging of the cytoskeleton, Nature Methods, 11, 731–733 (2014)