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US Patent granted for Super Resolution Localization Microscopy of naturally occurring GFP

No longer need for specifically designed switchable fluorescent dyes - The SPDMphymod technology of Prof. Christoph Cremer, University of Heidelberg & IMB Mainz, Germany, allows the use of standard, not genetically modified fluorescent proteins and of non-protein conventional fluorescent dyes in 2D & 3D super resolution microscopy.
"The granted US patent for the use of conventional fluorescent dyes is a strong unique selling point for our super resolution microscopy and a substantial market advantage in comparison with related methods which work with specially constructed photo-switchable or photo-activatable fluorescent dyes, or with typically toxic, special chemical environments", according to Dr. Andrea Nestl, innovation manager of the Technology Licensing Office (TLB), and responsible for the patent strategy, marketing and commercialization of this portfolio. ”This is a remarkable increase in value for our patent portfolio, combining 2D & 3D localization microscopy and structured illumination and additionally molecular biology application patents."

Fundamental to SPDMphymod (physically modifiable fluorophores) are blinking phenomena. The fluorescent molecules emit the same spectral light frequency, but with different spectral signatures based on the flashing characteristics. Conventional, well established and inexpensive fluorescent dyes like naturally occurring GFP and its derivatives like RFP, YFP, as well as Alexa, Atto and Cy2, Cy3 dyes can thus be used without any additional modification, in connection with standard embedding media or even physiological living cell conditions. Two or three conventional dyes can be detected in the co-localization mode, either as fluorescent fusion proteins or fluorescent labeled antibodies or combinations thereof.

Knowing that this will revolutionize super resolution light microscopy, because there is a multitude of material for investigation readily available for use without any additional preparation techniques, simply in the way as it is normally done by employing a standard confocal fluorescence or epifluorescence microscope, the inventors filed the SPDMphymod patent application on March 19th 2008, and subsequently published the scientific results in May 2008 by Reymann et al., and in September 2008 by Lemmer et al.

Since then, the Cremer lab has steadily increased the scope of applications of SPDMphymod, from the cell nucleus to nuclear membranes, cytoplasmic structures,clinically important cell membrane receptors, and to the analysis of cell junction complexes (Cremer et al. 2011 Biotechnology J. 6; Kaufmann et al. PLoSOne 2012).
By using visible laser light, this means that not only large cell areas can be studied two-dimensionally, but also cell complexes with a spatial resolution of every detail down to the range of few nanometer in 2D and up to a density of 2,8 • 10000/μm² within an area of up to 5000 µm² or few tens of nm in the 3D mode.

The availability of naturally occurring GFP (green fluorescent protein from jellyfish) and its derivatives has thoroughly redefined fluorescence microscopy and the way it is used in cell biology and other biological disciplines – the use in super resolution microscopy will start a new investigational era. Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien were awarded the 2008 Nobel Prize in Chemistry for their discovery and development of the green fluorescent protein.

The issue of this US patent for SPDMphymod is based on the SPDM (Spectral Precision Distance Microscopy) patent family, the first described farfield based localization microscopy technology (since the mid 1990s) that achieves an effective optical resolution several and even many times better than the conventional optical resolution, represented by the half-width of the main maximum of the effective point spread function.

GFP / RFP Dual color localization microscopy SPDMphymod / super resolution microscopy in a nucleus of a bone cancer cell: counting of 70,000 RFP-H2A-histone molecules & 50,000 GPF-Snf2H chromatin remodeling proteins (field of view of 470 µm², optical depth of 600 nm, each ‚spot‘ represents a single molecule, total 120000)
Foto: Prof. Christoph Cremer

The methodological simplicity of SPDMphymod technology is based on the fact that a single laser wavelength of suitable intensity is sufficient for nanoimaging the distribution of a given type of molecules, in contrast to other localization microscopy technologies which typically need two laser wavelengths in combination with especially designed photo-switchable/photo-activatable molecules and/or special chemical environments.

It is crucial for SPDMphymod that a single molecule is first transferred into a very long-living reversible dark state (with half-life of several seconds to minutes, i.e. orders of magnitude longer than typical ground state – triplet transitions), from which it returns to a fluorescent state, emitting many thousands of photons within several tens of milliseconds before returning into a very long-living so-called irreversible dark state.

"It is a substantial market advantage that there are manifold applications”, according to Dr. Andrea Nestl, responsible for the development of patenting, marketing and commercialisation strategy on behalf of the University of Heidelberg. The patent portfolio combining 2D & 3D localization microscopy, structured illumination, and additionally molecular biology applications will play an important role in the future in pharmaceutical, cell-biological, medical and biophysical research, i.e. wherever molecular ‘nano’imaging will be required on a cellular scale. For instance, hidden proteins or nucleic acids of a pharmacologically active 3D-molecule complex can be made visible without destroying the complex itself.

Christoph Cremer is Professor at the Kirchhoff Institute of Physics and the Institute of Pharmacy and Molecular Biotechnology (IPMB), both Institutes at the University of Heidelberg. Since 2011 he is also group leader in the field of Super Resolution Microscopy at the Institute of Molecular Biology gGmbH (IMB) in Mainz, Germany. In addition, he is a scientific member of the US-American Jackson Laboratory in Bar Harbor / Maine. Cremer is the appointed representative of the University of Heidelberg at the German Association of University Professors and Lecturers (Deutscher Hochschulverband DHV) and for many years he has been member of the The Senate, the most important decision-making body of the University of Heidelberg; from 2006 to 2009 he has been its Second Speaker.

P. Lemmer, M.Gunkel, D.Baddeley, R. Kaufmann, A. Urich, Y. Weiland, J.Reymann, P. Müller, M. Hausmann, C. Cremer (2008) SPDM – Light Microscopy with Single Molecule Resolution at the Nanoscale. Applied Physics B 93: 1-12.
J. Reymann, D. Baddeley, P. Lemmer, W. Stadter, T. Jegou, K. Rippe, C. Cremer, U. Birk (2008) High precision structural analysis of subnuclear complexes in fixed and live cells via Spatially Modulated Illumination (SMI) microscopy. Chromosome Research 16: 367 –382.
Manuel Gunkel, Fabian Erdel, Karsten Rippe, Paul Lemmer, Rainer Kaufmann, Christoph Hörmann, Roman Amberger and Christoph Cremer (2009): Dual color localization microscopy of cellular nanostructures. In: Biotechnology Journal 4, 927-938.

Kaufmann R, Piontek J, Grüll F, Kirchgessner M, Rossa J, Wolburg H, Blasig IE and Cremer C (2012). Visualization and quantitative analysis of reconstituted tight junctions using localization microscopy. PLoS One, 7, e31128.

Dr. Regina Kratt | idw
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