Localization Microscopy: Molecular Galaxies of protein transport in human blood-brain-barrier, tight junctions networks, gene transcription, single molecules, nuclear histones, viruses

1. January 1, 2013
Letizia Mancino
HD MZ
Localization Microscopy:
“Molecular Galaxies 2013“
C. Cremer
Institute of Molecular Biology (IMB), D-55128 Mainz
Institute of Pharmacy and Molecular Biotechnology (IPMB)/Kirchhoff
Institute of Physics (KIP), D-69120 University Heidelberg
www.imb-mainz.de.

2. Molecular Galaxies
Conventional Localization of Protein Transport
Microscopy
in a Human Blood-
Brain Barrier Model
O. Huber, A. Brunner, P.
Maier, R. Kaufmann, P.-O.
Couraud, C. Cremer, G.
Fricker (2012)

3. Molecular Galaxies of Tight Junction
Networks formed by individual Claudin
Molecules in Human Cells
Conventional Microscopy Localization Microcopy
R. Kaufmann, J.Piontek, F. Grüll, M. Kirchgessner, J. Rossa, H.
Wolburg, I. E. Blasig, C. Cremer (2012)

4. Molecular Galaxies of Gene Transcription
Conventional Microscopy Localization Microscopy
Positions
R. Kaufmann ,
C.Cremer,
J.Gall
(2012)

5. Deep Insights
into the Interior
of Molecular
Galaxies:
Single
Molecules
Single Molecules Nuclear Histones a) on Surface
b) Inside Cell
300 nm Nucleus
c),d) in Virus
Single Viruses C. Cremer (2012)

6. Principle of Spectrally Assigned Localization Microscopy
(SPDM, Spatial Position Determination Microscopy): Optical
Isolation of Diffraction Images of individual Molecules:
Determination of Gravity Centers
3 point-like objects in x,y plane with
next neigbour distances 50 nm
a) Labelling with same spectral
signature
b) Labeling with different unique
spectral signatures B,G,R
Computation (scalar Theory):
NA = 1.4, λexc = 488 nm
Optical Isolation by the
“Supernova“ Mode: Induction of
stochastically distributed short
flashes of light emission by
Cremer et al. 1996, 1999, 2010, 2012 individual molecules

7. Present State of Localization Microscopy (wide field)
realized @ C. Cremer Lab (January 1, 2013)
- Best Optical Resolution OR (resolvable distance):
OR ~ 5 nm (~ 1/100 λexc, from localization precision)
- Structural Resolution (imaging capability):
● Mean (2D) distance between individual molecules actually
detected: ~ 6 nm ( ~ 1/80 λexc)
● Density of individually detected molecules : ~ 2,8 · 104/µm2
● 3D single molecule resolution inside cells:
ca. 20 nm laterally, 30 – 50 nm axially
● Optical Sectioning: ~ 15 nm

8. Publications Cremer-Lab 2012 part I
C. Cremer, Optics far Beyond the Diffraction Limit: From Focused
Nanoscopy to Spectrally Assigned Localization Microscopy
(2012). In: Springer Handbook of Lasers and Optics, 2nd edition
(F. Träger, Edit.), pp. 1351 – 1389.
T. Cremer, Y. Markaki, B. Hübner, A. Zunhammer, H. Strickfaden,
S. Beichmanis, M. Heß, L. Schermelleh, M. Cremer, C. Cremer
(2012) Chromosome Territory Organization within the Nucleus. In:
Encyclopedia of Molecular Cell Biology and Molecular Medicine:
Epigenetic Regulation and Epigenomics, 2nd Edition (R.A. Meyers,
Edit.), pp. 1-30.Wiley-VCH.
R. Kaufmann, C. Cremer, J. G. Gall (2012a) Superresolution
imaging of transcription units on newt lampbrush chromosomes.
Chromosome Research, DOI 10.1007/s10577-012-9306-z-

9. Publications Cremer-Lab 2012 part II
R. Kaufmann, J. Piontek, F. Grüll, M. Kirchgessner, J.Rossa, H.
Wolburg, I. E. Blasig, C. Cremer (2012b) Visualization and
quantitative analysis of reconstituted tight junctions using
localization microscopy. PLoS One 7 (2 ) e31128: 1 – 9.
O. Huber, A. Brunner, P. Maier, R. Kaufmann, P.-O. Couraud, C.
Cremer, G. Fricker (2012) Localization microscopy (SPDM)
reveals clustered formations of P-Glycoprotein in a human blood-
brain barrier model. PLoS ONE 7 (9) e44776: 1-10.
T. Ach, G. Best, S. Rossberger, R. Heintzmann, C. Cremer, S.
Dithmar (2012) Autofluorescence imaging of human RPE cell
granules using structured illumination microscopy. Br. J.
Ophthalmology, DOI 10.1136/bjophthalmol-2012-301547.