Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:


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
Further information:

More articles from Life Sciences:

nachricht Rainbow colors reveal cell history: Uncovering β-cell heterogeneity
22.09.2017 | DFG-Forschungszentrum für Regenerative Therapien TU Dresden

nachricht The pyrenoid is a carbon-fixing liquid droplet
22.09.2017 | Max-Planck-Institut für Biochemie

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: The pyrenoid is a carbon-fixing liquid droplet

Plants and algae use the enzyme Rubisco to fix carbon dioxide, removing it from the atmosphere and converting it into biomass. Algae have figured out a way to increase the efficiency of carbon fixation. They gather most of their Rubisco into a ball-shaped microcompartment called the pyrenoid, which they flood with a high local concentration of carbon dioxide. A team of scientists at Princeton University, the Carnegie Institution for Science, Stanford University and the Max Plank Institute of Biochemistry have unravelled the mysteries of how the pyrenoid is assembled. These insights can help to engineer crops that remove more carbon dioxide from the atmosphere while producing more food.

A warming planet

Im Focus: Highly precise wiring in the Cerebral Cortex

Our brains house extremely complex neuronal circuits, whose detailed structures are still largely unknown. This is especially true for the so-called cerebral cortex of mammals, where among other things vision, thoughts or spatial orientation are being computed. Here the rules by which nerve cells are connected to each other are only partly understood. A team of scientists around Moritz Helmstaedter at the Frankfiurt Max Planck Institute for Brain Research and Helene Schmidt (Humboldt University in Berlin) have now discovered a surprisingly precise nerve cell connectivity pattern in the part of the cerebral cortex that is responsible for orienting the individual animal or human in space.

The researchers report online in Nature (Schmidt et al., 2017. Axonal synapse sorting in medial entorhinal cortex, DOI: 10.1038/nature24005) that synapses in...

Im Focus: Tiny lasers from a gallery of whispers

New technique promises tunable laser devices

Whispering gallery mode (WGM) resonators are used to make tiny micro-lasers, sensors, switches, routers and other devices. These tiny structures rely on a...

Im Focus: Ultrafast snapshots of relaxing electrons in solids

Using ultrafast flashes of laser and x-ray radiation, scientists at the Max Planck Institute of Quantum Optics (Garching, Germany) took snapshots of the briefest electron motion inside a solid material to date. The electron motion lasted only 750 billionths of the billionth of a second before it fainted, setting a new record of human capability to capture ultrafast processes inside solids!

When x-rays shine onto solid materials or large molecules, an electron is pushed away from its original place near the nucleus of the atom, leaving a hole...

Im Focus: Quantum Sensors Decipher Magnetic Ordering in a New Semiconducting Material

For the first time, physicists have successfully imaged spiral magnetic ordering in a multiferroic material. These materials are considered highly promising candidates for future data storage media. The researchers were able to prove their findings using unique quantum sensors that were developed at Basel University and that can analyze electromagnetic fields on the nanometer scale. The results – obtained by scientists from the University of Basel’s Department of Physics, the Swiss Nanoscience Institute, the University of Montpellier and several laboratories from University Paris-Saclay – were recently published in the journal Nature.

Multiferroics are materials that simultaneously react to electric and magnetic fields. These two properties are rarely found together, and their combined...

All Focus news of the innovation-report >>>



Event News

“Lasers in Composites Symposium” in Aachen – from Science to Application

19.09.2017 | Event News

I-ESA 2018 – Call for Papers

12.09.2017 | Event News

EMBO at Basel Life, a new conference on current and emerging life science research

06.09.2017 | Event News

Latest News

Rainbow colors reveal cell history: Uncovering β-cell heterogeneity

22.09.2017 | Life Sciences

Penn first in world to treat patient with new radiation technology

22.09.2017 | Medical Engineering

Calculating quietness

22.09.2017 | Physics and Astronomy

More VideoLinks >>>