Christoph Cremer and Thomas Cremer 40 years of joint ... - TLB

Christoph Cremer and Thomas Cremer
40 years of joint research to explore the functional nuclear
architecture
Christoph Cremer
From laser-uv-microbeam irradiation to super-resolution fluorescence
Microscopy
Institute of Molecular Biology (IMB), D-55128 Mainz, Germany, and Kirch hoff Institute of
Physics (KIP),University Heidelberg, D-69120 Heidelberg, Germany e-mail: c. cremer@imbmainz.
de
For about a century, far-field light microscopy suffered from the optical resolution
restriction imposed by the diffraction barrier (ca. 200 nm laterally, 600 nm along the
optical axis). Here, I point out our conceptual and technical contributions, which have led
from the development of laser-uv-microbeams to super-resolution far field microscopy
with emphasis on applications to study nuclear architecture.
In a patent application filed in 1971 we (C. and T. Cremer) proposed the possibility of
a "HoFo" (Holographic Focusing) laser scanning microscope based on the idea that laser
illumination from all sides (4π-geometry) should allow the generation of a constructive
focus with a considerably smaller diameter compared to focusing with a single
conventional lens.
In addition to applications in super-resolution fluorescence microscopy we proposed that
holographic focusing scanning microscopes might become useful for high capacity
storage devices with array elements, whose optical behavior can be changed in a
reversible or irreversible way by appropriate intensities and frequencies of the
photoswitching electromagnetic field.
Recent calculations based on electromagnetic theory and conservative assumptions
indicate that constructive interference of multiple point-like light sources, spatially
distributed in a "4TT" mode, should provide a 3D resolution improved by a factor of
~25 compared to conventional confocal microscopy.
Such a resolution would correspond to values presently realized in commercial "focused
nanoscopy" microscopes with the additional advantage of allowing large working
distances. The ultimate theoretical limit of holographic "4TT" focusing (including novel
possibilities of attosecond laser physics) deserves further consideration.
Based on experience gained with the construction of a laser-uv-microbeam for 257 nm
excitation (1974), we described in 1978 the first detailed construction plan for a detection
pinhole based laser scanning fluorescence microscope to obtain highly
resolved, threedimensional (3D) images of cellular structures.
In the early 1990ies I was involved in the development of 4Pi laser microscopy based on
constructive focusing of laser light through two opposing regular objective lenses.
With members of my group I filed patent applications in 1996/1997 (European and USA
patents granted in 2001/2002) on a procedure for superresolution microscopy by
multispectral precision distance measurements (localization microscopy) in biological
micro-objects.

It is based either on using fluorophores with different emission spectra or on fluorophores,
which have the same emission spectrum, but different time dependent emission
characteristics, such as fluorescence lifetime, luminescence and phosphorescence.
Generally, such differences in "spectral signature" meant any photophysical property
which can be used for optically discriminated registration. For this procedure the
Abbe/Rayleigh limit of the nearest resolvable distance of two point-like targets is no
longer valid.
This approach has subsequently been developed and applied in my group for the 3D
analysis of the molecular nanostructure of the genome in the mammalian cell nucleus.
In a collaborative effort, a microscope procedure employing the time domain (in this
case fluorescence lifetime of single molecules) was realized in 2002.
Since the mid 1990ies, my group developed further methods to realize an enhancement
in spatial resolution by spatially modulated illumination (SMI) microscopy (1997) and
by structured excitation illumination microscopy using a diffraction grating (1999). In a
patent application filed in 2001 (3D LIMON), we described the combination of SMI
microscopy and localization microscopy SPDM to analyze objects of subwavelength size,
making use of random labeling procedures for optical object reconstruction.
Based on our experience with the development of multispectral distance microscopy we
built a spectral position determination (SPDMphymod) epifluorescence microscope again
based on the time domain, in this case employing the blinking properties of standard
fluorophores, such as fluorescent proteins like GFP and Alexa dyes (2008).
Today, these approaches allow a manyfold improved optical resolution, our groups
currently studying the nuclear topography of nucleosomes and RNA Pol II.
For Reviews see:
Cremer C, Cremer T (1971) 4Π Punkthologramme: Physikalische Grundlagen und mögliche
Anwendungen. Enclosure to Patent application DE 2116521 „Verfahren zur Darstellung bzw. Modifikation von
Objekt-Details, deren Abmessungen außerhalb der sichtbaren Wellenlängen liegen" (Procedure for the
imaging and modification of object details with dimensions beyond the visible wavelengths). Filed April 5,
1971; publication date October 12, 1972.Deutsches Patentamt, Berlin.
Rouquette J, Cremer C, Cremer T, Fakan S (2010) Functional nuclear architecture studied by microscopy:
Present and future. Int. Rev. Cel. Mol. Bio. 282: 1-156.
Brunner A, Best G, Lemmer P, Ambergr R, Ach T, Dithmar S, Heintzmann R, Cremer C (2011)
Fluorescence microscopy with structured excitation illumination. In: Handbook of Biomedical Optics.
Editors: Boas DA, Pitris C, Ramanujam N, CRC Press, Taylor and Francis Group, London, pp. 543-560.
Cremer C, Kaufmann RY, Müller P, Ruckelshausen T, Lemmer P, Geiger F, Degenhard S, Wege C,
Lemmermann NA, Holtappels R, Strickfaden H, Hausmann M. (2011) Superresolution imaging of
biological nanostructures by spectral precision distance microscopy. Biotechnol. J. 6: 1037-1051.