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Super Resolution Light Microscopy Super Resolution Light Microscopy Wladimir Schaufler Nobel Prizes for Microscopy Developments Richard Zsigmondy invented the ultramicroscope. Nobel Price 1925 Frits Zernike invented the phase-contrast microscope. Nobel Price 1953 Maria Goeppert-Mayer described the two-photon excitation fluorescence. Nobel Price 1963 Ernst Ruska build the first electron microscope. Nobel Price 1986 Gerd Binnig designed the scanning tunneling microscope (STM). Nobel Price 1986 Nobel Prizes for using Microscopy as a Primary Scientific Instrument More than 10 Nobel Prizes, especially in the last 10 years including Harald zur Hausen, Nobel Prize for Physiology or Medicine, 2008 (1983 to 2003 DKFZ chairman). 11/20/2013 | Page 2 Wladimir Schaufler Super Resolution Light Microscopy Scale Issue 11/20/2013 | Page 3 Wladimir Schaufler Super Resolution Light Microscopy Optical Resolution (lateral) for Light Microscopy: Abbe Limit Numerical Aperture = n ·sin α Today best available objective: NA = 1.49 Source: Olympus Source: Abbe School of Photonics, Jena d 0,34 21,49 λ(Red Light) = 600nm ~210 nm Resolution Limit 11/20/2013 | Page 4 Wladimir Schaufler Super Resolution Light Microscopy Spatial Resolution of Biological Imaging Techniques http://zeiss-campus.magnet.fsu.edu/articles/superresolution/palm/practicalaspects.html 11/20/2013 | Page 5 Wladimir Schaufler Super Resolution Light Microscopy Localization Microscopy 11/20/2013 | Page 6 Wladimir Schaufler Super Resolution Light Microscopy Significantly Simplified Optical Setup for Localization Microscopy High Laser Power Density in Sample UV 405nm Blue 491nm Green 561nm Red 642nm DKFZ Super Resolution Laboratory (was established by W. Schaufler) at Central Microscopy Facility (Head: Dr. Felix Bestvater) 11/20/2013 | Page 7 Wladimir Schaufler Super Resolution Light Microscopy Localization Microscopy (SPDM) (developed and patented in the Prof. Dr. Dr. Christoph Cremer Group) Sven Beichmanis X protein labeled with alexa488 fluorescence marker, 50ms per frame 11/20/2013 | Page 8 Wladimir Schaufler Super Resolution Light Microscopy Fitting Single-Molecule Pixel Data to a Gaussian Function 1) Amplitude(Photons), 2) x(nm), 3) y(nm), 4) Loc.Error+_x(nm), 5) Loc.Error+_y(nm), 6) FWHM*_x(nm), 7) FWHM*_y(nm), 8) Photonamount (Integral), http://zeiss- campus.magnet.fsu.edu/articles/superresolution/palm/practicalaspects.html 9) Framenumber 10) Asymetry dx/dy *FWHM (Full Width at Half Maximum) ~ 200nm +Localization Error ~ 5-25nm Table with Data for Each Single Fluorescence Labeled Molecule (Matlab) 11/20/2013 | Page 9 Wladimir Schaufler Super Resolution Light Microscopy Conventional Microscopy Gunkel et al. 2009 Optical Section View 11/20/2013 | Page 10 Wladimir Schaufler Super Resolution Light Microscopy Localization Microscopy (SPDM) Analysis of Histone- Chromatin Remodelling Factor Distribution Gunkel et al. 2009 Optical Section View 11/20/2013 | Page 11 Wladimir Schaufler Super Resolution Light Microscopy microRNAs promote metastasis Our protocol Seed of cultured cells on coverslips Labeling of metastasis-specific miRNA/s (transfection or FISH) Conventional microscopy Exosome with miR Super Resolution Vesicle free miRs Microscopy SPDM 3D-recostruction&quantative Agronaute- ptotein analyse of spatial distribution and localization (home-made software) We work here! microRNA Cancer cell Nearly no data are available about miRNA distribution within cells or tissues Huang et al, 2010 Provided by Dr. Oleksiuk, DKFZ 11/20/2013 | Page 12 Wladimir Schaufler Super Resolution Light Microscopy Acknowledgements Prof. Dr. Dr. Christoph Cremer Group • Margund Bach • Dr. Manuel Gunkel • Sebastian Pres • Kirti Prakash • Gerrit Best • Martin Hagmann • Sabrina • Aleksander • Dr. Udo Birk • Florian Schock Rossberger Szczurek DKFZ Microscopy Facility • Dr. Felix Bestvater (Head) • Dr. Damir Krunic • Manuela Brom • Dipl. Ing. Heinz Eipel DKFZ Clinical Cooperation Unit Molecular Oncology of Solid Tumors • Prof. Dr. Dr. Heike Allgayer (Head) • Dr. Olga Oleksiuk • Kerem Tezcan 11/20/2013 | Page 13 Wladimir Schaufler Super Resolution Light Microscopy.
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