Professor Christoph Cremer of the University of Heidelberg‘s
Kirchhoff-Institute of Physics meets the
true needs of researchers in the fields of molecular biology and medicine with
the world’s fastest super resolution microscope Vertico SMI, i.e. the ability
to use conventional fluorescent dyes such as GFP when investigating clusters of
living cells. The methods developed by
Professor Cremer are eliciting great interest in Europe and the USA, but also
in many other countries including China and Japan. For the
commercialisation of his nanoimaging procedures, Professor Cremer
collaborates with Dr Andrea Nestl, Innovation Manager of the
Technologie-Lizenz-Büro (TLB) GmbH in Karlsruhe (both pictured above).
1. Research teams worldwide compete for the best view
into the molecular world of cells. How
are you, as a pioneer in the field of super resolution microscopy, setting new
standards? Professor Christoph Cremer: Our approach to super resolution microscopy combines
localization microscopy SPDM and spatially modulated illumination microscopy
SMI which allows us to work with the usual, well established fluorescent
dyes. The most important ones are of
course natural fluorescent proteins such as GFP, synthetic dyes such as Alexa fluorescent
dyes 488, 568, 647 and fluorescein which is used in ophthalmology for the
diagnosis of corneal diseases.
Using our wide field microscope Vertico-SMI we are
even able to investigate several even living cells simultaneously at the
molecular level. We achieve resolutions
of 10 nm in 2D and 40 nm in 3D using visible laser light. We have special software protected by patent
rights which allows us to achieve extremely fast image recording and processing
speeds. Print ready pictures are thus
available within a few minutes following the recording of the image.
2. The Nobel Prize winning fluorescence molecule GFP
plays an outstanding role in research experiments investigating cells. What exactly does it mean for researchers
that your nanoscope works with the same fluorescence molecule while producing
the best resolution achievable at present? Professor Christoph Cremer: These marker techniques are
used in thousands of biomedical laboratories all around the world to study cell
biology and a huge number of such preparations are in existence. These preparations are however not suitable
for the recording of nanoscope images using commercially available super
resolution microscopes. They would first
have to be reproduced using expensive switchable dyes.
With our Vertico SMI it is
however for the first time possible to continue working with the usual GFP
fluorescent dye group. It also works
with other unmodified fluorescent dyes in this group, such as RFP and YFP. Particularly exciting is the co-localization
method 2CLM (2Colour Localization Microscopy) which we are also able to apply
at the level of single molecules.
3. To be able to recognize the tiny structures within
a cell it is necessary to „turn the light on“ using light molecules. What is it that is so clever about your
method using GFP? Professor Christoph Cremer:
The trick is that we use a characteristic of GFP and other fluorescent molecule
that to date was seen as an annoying interference. If GFP molecules are excited with a laser
light, each molecule reacts by emitting a single flash of light within two
minutes –not simultaneously but distributed over that period of time.
Using our localization microscopy SPDMphymod, we can
record thousands of images of ever changing light constellations during the
above-mentioned two minute period. It is
from these images that we can compute the high resolution cartographic
image. It is also helpful that because
of this flashing phenomenon we can get away with a single wave length laser,
unlike when using switchable or photon activated fluorescent dyes.
4. To be able to count molecules in cells in such
a simple way has the potential to revolutionize the whole molecular biology,
medical and pharmaceutical research.
What new break-throughs can be expected from the application of Cremer’s
nanoscopy in these fields? Professor Christoph Cremer:
Through a diverse range of collaborative work we are developing totally new
strategies for the prevention, risk reduction and therapy of diseases. Working with cardiologists we are
investigating at the nanoscopic level ion channels which play an important role
in the regulation of the heart rhythm.
This is aimed at improving the pharmaceuticals available to prevent
heart attacks.
In other cooperative
projects we hope to understand better and learn to influence the mechanisms
that are at work at the blood brain barrier so that pharmaceuticals may be
developed that are better able to pass through this barrier – this would be of
great benefit to patients with brain tumors or those with severe psychological
problems. In our virology-focused
cooperative work we are often able to analyze the docking process between virus
and cell membrane, which are the foundation of the infectious process, much
more quickly and simply than using conventional electron microscopy. This provides a substantial advantage in the
development of new pharmaceuticals against viral infections such as the flu or
AIDS. At present, we are also
considering using our nanoscopy in the field of material sciences.
5. The development of high tech microscopes is
very expensive. What are the conditions
that allowed you to develop your super resolution microscope? Professor Christoph Cremer:
Decades of investments were necessary for our development. The first patent application in 1971, which
covered the concept of the 4Pi microscopy and stems from the time of my diploma
work, was financed by my family.
The
further development since 1970 was supported on an ongoing basis by the Deutsche
Forschungsgemeinschaft (DFG). In
addition, financial support was provided by the German Ministry for Education
and Research, the European Union in the context of a consortium on molecular
imaging and two research support programs by the State Baden-Wuerttemberg as
well as PhD scholarships by the State Baden-Wuerttemberg and the DFG. The University of Heidelberg has for many
decades provided the infrastructure, such as the laboratory space and
equipment, as well as human resources.
6. It is not easy to understand the field of
light optical nanoscopy, given that there are four super resolution microscopes
either already on the market or being prepared to be launched
commercially. How is your technology
different from what is offered by others and how do you gauge its market
potential?
Professor Christoph Cremer:
Other super resolution microscopes such as ELYRA and N-STORM can only be used
in conjunction with photo-activated fluorescence molecules and do not work with
standard fluorochromes. This also brings
with it the disadvantage of a substantially slower imaging speed so that it is
not possible to take images of living cells with high molecule densities. Other nanoscopes such as STED are based on
confocal light microscopy and can therefore only capture quickly a small
area. A difference with the multicolor
3D-SIM microscopy such as OMX is the much higher resolution, in fact more than
double what we can achieve with two colors in 3D.
Our Vertico-SMI surpasses the other comparable technologies
in that it offers all the relevant features and, in addition, our rapid
software provides a quick solution to the complex image processing, allowing
on-line nanoimaging.
We believe that our market outlook is extremely
positive. Because our Vertico-SMI can be tailored to suit particular
applications and thus its complexity and therefore its price is scalable. The price for certain applications will
therefore be substantially less than the cost of currently available super
resolution nanoscopes, which is in the vicinity of US$ 1.5 million.
7. In your research career you have more than
once broken through the limits of what was possible in optical microscopy. What were the milestones and where lies your
next goal? Professor Christoph Cremer:Following the patent application covering the 4Pi laser scanning
microscope in
1971 mentioned earlier, I developed the first laser UV micro
irradiation
technique which allowed the targeted introduction of DNA damage in
surviving
cells which made it possible to investigate the functional genome
structure. The next step came in 1978
with the concept of the confocal laser scanning microscope (CLSM) for
the
investigation of fluorescent objects. This
technology can nowadays be found in almost every molecular biology
institute. Unfortunately, my brother and I didn’t seek
patent protection for this invention and it was subsequently realized
by
several research groups and companies.
It was in the early 1990s that I began with the development of the base
technology for my current super resolution microscope. Recently
we have submitted a new paper which
describes a further breakthrough.
Since winning the Bwcon-Business-Award of the
Heidelberger Innovationsforum, I am actively pushing ahead with the
commercialisation of this technology. In
this endeavour I am supported professionally by the Technologie-Lizenz-Büro der
Baden-Württembergischen Hochschulen, which also manages my patent portfolio on
behalf of the University of Heidelberg.
Thank you for talking with us.
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