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.

January 1, 2013

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

Conventional Localization Microscopy

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)

Positions

Conventional Microscopy Localization Microscopy

Molecular Galaxies of Gene Transcription

R. Kaufmann , C.Cremer, J.Gall (2012)

Single Molecules Nuclear Histones Single Viruses

300 nm

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

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

individual molecules Cremer et al. 1996, 1999, 2010, 2012

Principle of Spectrally Assigned Localization Microscopy (SPDM, Spatial Position Determination Microscopy): Optical Isolation of Diffraction Images of individual Molecules: Determination of Gravity Centers

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

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-

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.